Nanomanipulation coupled nanospray mass spectrometry (NMS)

ABSTRACT

A coupled nanomanipulation and nanospray mass spectrometry (NMS) system for single cell, single organelle, and ultra-trace molecular analysis is disclosed herein. The system primarily comprises a bio-workstation coupled to a NMS. The bio-workstation primarily comprises of a nanomanipulator stage with a plurality of nano-positioners attached to a cabinet with a piezo voltage source and a pressure injector. The present invention further describes a fingerprint lift method that when coupled with the system disclosed herein can be used for retrieval and analysis of trace amounts of drug and explosive residues. The system described herein has been used in the areas of trace and document analysis within the forensic field, trace fiber analysis, and electrostatic lifts for illicit drugs, as well as document and painting analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application of U.S. ProvisionalPatent Application No. 61/391,842 filed on Oct. 11, 2010, and entitled“Nanomanipulation Coupled Nanospray Mass Spectrometry (NMS)” which ishereby incorporated by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of massspectrometry (MS), and more particularly to a technique for single cell,single organelle, and ultra-trace molecular analysis usingnanomanipulation coupled with nanospray mass spectrometry (NMS).

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

REFERENCE TO A SEQUENCE LISTING

None.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with mass spectrometry techniques and methods.

U.S. Patent Application Publication No. 2009/0261244 (Syms, 2009)provides a method of aligning a nanospray capillary needle, a set ofelectrodes, and a capillary input to a mass spectrometer. The electrodesystem is formed using microengineering technologies, as an assembly oftwo separate chips. Each chip is formed on an insulating plasticsubstrate. The first chip carries mechanical alignment features for thecapillary electrospray needle and the API mass spectrometer input,together with a set of partial electrodes. The second chip carries a setof partial electrodes. The complete electrode system is formed when thechips are assembled in a stacked configuration, and consists of aneinzel lens capable of initiating a Taylor cone and separating ions fromneutrals by focusing.

U.S. Pat. No. 7,385,189 (Goodley et al. 2008) provides an apparatus andmethod for use with a mass spectrometry system. The invention providesan ion source for providing radiative heating to an ionization region.The ion source includes a nanospray ionization device for producing ionsand a conduit adjacent to the ionization device for receiving ions fromthe ionization device. The conduit includes a conductive material for,providing indirect radiative heating to the ionization region. Directradiative heating may also be provided using a heater in the conduit.The ion source may be used separately or in conjunction with the massspectrometry system. When used in conjunction with a mass spectrometrysystem a detector may also be employed down stream from the device. Amethod for desolvating an analyte using the device is also disclosed.

U.S. Pat. No. 6,812,460 (Stallcup and Baur, 2004) discloses a method ofnano-manipulation, including providing a nano-scale object movablypositioned over a substrate and positioning a probe of a scanning probemicroscope proximate the nano-scale object. The probe is then movedacross the substrate along a gyrating path proximate the nano-scaleobject to reposition the nano-scale object.

SUMMARY OF THE INVENTION

The present invention describes a technique that combinesnanomanipulation and nanospray mass spectrometry (NMS) for the analysisof a single cell, a single organelle, and ultra-trace amounts of one ormore molecules.

The present invention in one embodiment provides a method foridentifying, detecting, analyzing or combinations thereof of one or moreanalytes from a substrate comprising the steps of: (i) providing thesubstrate comprising the one or more analytes to be detected, whereinthe substrate is mounted directly or indirectly on an invertedmicroscopic stage, (ii) providing an extraction system for an extractionof the one or more analytes from the substrate followed by a transfer toa detection system, wherein the detection system is coupled to theextraction system, wherein the extraction system comprises: a) aworkstation comprising a plurality of moveable nanopositioners, whereinthe nanopositioners are capable of movement in a X-Y-Z plane, whereinthe nanopositioners hold one or more probes, grippers, capillary tips orany suitable accessory for an extraction and transfer of a liquid phase,b) a pressure injector for delivery of a pressurized extraction solventthrough the capillary tip, wherein the capillary tip is placed in closeproximity to the substrate, c) a voltage source, d) a joystick or adigital controller for controlling the movement of the nanopositioners,and e) a mass spectrometer that is offline or is connected to theextraction system for receiving the one or more dissolved analytestransferred by the extraction system, (iii) dissolving the one or moreanalytes by an injection of the extraction solvent onto the substrate,(iv) aspirating the dissolved analytes by the one or more capillarytips, (v) injecting the aspirated analytes into the mass spectrometer toobtain a mass spectrum, and (vi) identifying the one or more analytes bya m/z ratio in the mass spectrum. Additionally, the method furthercomprises the step of determining a concentration of the one or moreanalytes in the substrate.

In one aspect of the method disclosed hereinabove the one or moreanalytes are selected from the group consisting of industrial, chemical,and biological materials, biological cells, paints, inks, pigments,dyes, illicit drugs, pharmaceuticals, proteins, and combinations andmodifications thereof. In another aspect the substrate is selected fromthe group consisting of fibers, clothing, hair, paper, money, computerchips, treated wood, laminate, metal, manipulated surfaces includingmylar electrostatic lift films, plastic, and combinations andmodifications thereof. In a specific aspect of the method the massspectrometer is equipped with a nanospray source. In another aspect thesubstrate is mounted on a non-inverted microscopic stage.

In another aspect the method is used for analysis of documents, analysisof artworks, illicit drug detection, forensic and toxicology studies,and combinations and modifications thereof. In yet another aspect theextraction solvent comprises water, polar organic and inorganicsolvents, mixtures of polar and non-polar solvents, and combinations andmodifications thereof.

In another embodiment the instant invention discloses a system foridentifying, detecting, analyzing or combinations thereof of one or moreanalytes from a substrate comprising: an inverted or non-invertedmicroscopic stage capable of holding the substrate comprising the one ormore analytes, a workstation comprising a plurality of moveablenanopositioners, wherein the nanopositioners are capable of movement ina X-Y-Z plane, wherein the nanopositioners hold one or more probes,grippers, capillary tips or any suitable accessory for an extraction andtransfer of a liquid phase, a pressure injector for delivery of apressurized extraction solvent through the capillary tip, wherein thecapillary tip is placed in close proximity to the substrate, a voltagesource, a joystick or a digital controller for controlling the movementof the nanopositioners, and a mass spectrometer that is offline or isconnected to the extraction system for receiving the one or moredissolved analytes transferred by the extraction system and foridentifying the one or more analytes by a m/z ratio from a generatedmass spectrum. The system disclosed above further comprises the step ofdetermining a concentration of the one or more analytes in thesubstrate. In one aspect the one or more analytes are selected from thegroup consisting of industrial, chemical, and biological materials,cells, organelles, paints, inks, pigments, dyes, illicit drugs,pharmaceuticals, proteins, and combinations and modifications thereof.

In specific aspects the illicit drug is cocaine, the organelle ismitochondria and the substrate is selected from the group consisting offibers, clothing, hair, paper, money, computer chips, treated wood,laminate, metal, manipulated surfaces including mylar electrostatic liftfilms, plastic, and combinations and modifications thereof. In a relatedaspect the mass spectrometer is equipped with a nanospray source. Inanother aspect the system is used for analysis of documents, analysis ofartworks, illicit drug detection, forensic and toxicology studies, andcombinations and modifications thereof. In yet another aspect theextraction solvent comprises water, polar organic and inorganicsolvents, mixtures of polar and non-polar solvents, and combinations andmodifications thereof. It will be understood that addition ofsignificant acid/base chemistry to the extraction solvent can modify theionization process.

Yet another embodiment of the instant invention relates to a method forcompositional analysis, concentrating one or more analytes orcombinations thereof comprising the steps of: a) providing the one ormore analytes in a first liquid phase mounted directly or indirectly onan inverted microscopic stage, b) providing an extraction system for anextraction of the one or more analytes from the substrate followed by atransfer to a detection system, wherein the detection system is coupledto the extraction system, wherein the extraction system comprises: (i) aworkstation comprising a plurality of moveable nanopositioners, whereinthe nanopositioners are capable of movement in a X-Y-Z plane, whereinthe nanopositioners hold one or more probes, grippers, capillary tips orany suitable accessory for an extraction and transfer of a liquid phase,(ii) a pressure injector for delivery of a pressurized extractionsolvent through the capillary tip, wherein the capillary tip is placedin close proximity to the substrate, wherein the extraction solvent isimmiscible or partially miscible with the first liquid phase, (iii) avoltage source, (iv) a joystick or a digital controller for controllingthe movement of the nanopositioners, and (v) a mass spectrometerconnected to the extraction system (online) or not directly connected tothe extraction system (off line) for receiving the one or more analytestransferred by the extraction system, c) solubilizing or partitioningthe one or more analytes between the first phase and the extractionsolvent by an injection of the extraction solvent onto the substrate, d)aspirating a mixture of the solubilized or partitioned analytes by theone or more capillary tips, e) mixing the aspirated mixture by amovement of the mixture in the capillary tube, f) injecting theaspirated analytes into the mass spectrometer to obtain a mass spectrum,and g) identifying the one or more analytes by a m/z ratio in the massspectrum. The method as disclosed herein further comprises the step ofdetermining a concentration of the one or more analytes in thesubstrate. In one aspect the step of determining the concentration isdone by an injection of an internal standard, wherein the internalstandard is a pure sample or a derivative of the analyte having asimilar mass. In another aspect the one or more analytes are selectedfrom the group consisting of biomolecules, lipophilic compounds, oils,triacylglycerols, vegetable oil products, cottonseed oil, serum,proteins, antibodies, and combinations and modifications thereof. Inspecific aspects the analyte is cottonseed oil or rabbit serum.

In another aspect the mass spectrometer is equipped with a nanospraysource. In yet another aspect the extraction solvent comprises water,polar organic and non-polar organic solvents, mixtures of polar andnon-polar solvents, and combinations and modifications thereof. It willbe understood that addition of significant acid/base chemistry to theextraction solvent can modify the ionization process. In another aspecta non-limiting example of an extraction solvent is a 1:1 mixture ofchloroform and methanol, comprising about 2% acetic acid. It will beunderstood by the skilled artisan that the choice of a solvent willdepend on the system to be extracted.

In one embodiment the present invention relates to a system forcompositional analysis, concentrating one or more analytes orcombinations thereof of one or more analytes from a substratecomprising: an inverted microscopic stage mounted with or capable ofholding the one or more analytes in a first liquid phase, an extractionsystem for an extraction of the one or more analytes from the substratefollowed by a transfer to a detection system, wherein the detectionsystem is coupled to the extraction system, wherein the extractionsystem comprises: a workstation comprising a plurality of moveablenanopositioners, wherein the nanopositioners are capable of movement ina X-Y-Z plane, wherein the nanopositioners hold one or more probes,grippers, capillary tips or any suitable accessory for an extraction andtransfer of a liquid phase, a pressure injector for delivery of apressurized extraction solvent through the capillary tip, wherein thecapillary tip is placed in close proximity to the substrate, wherein theextraction solvent is immiscible or partially miscible with the firstliquid phase, a voltage source, a joystick or a digital controller forcontrolling the movement of the nanopositioners, and a mass spectrometerconnected to the extraction system for receiving the one or moreanalytes transferred by the extraction system. In one aspect the systemcan be used to determine a concentration of the one or more analytes inthe substrate. In another aspect the step of determining theconcentration is done by an injection of an internal standard, whereinthe internal standard is a pure sample or a derivative of the analytehaving a similar mass. In another aspect the one or more analytes areselected from the group consisting of biomolecules, lipophiliccompounds, oils, triacylglycerols, vegetable oil products, cottonseedoil, serum, proteins, antibodies, and combinations and modificationsthereof.

In specific aspects the analyte is cottonseed oil or rabbit serum andthe mass spectrometer is equipped with a nanospray source. In anotheraspect the extraction solvent comprises water, polar organic andnon-polar organic solvents, mixtures of polar and non-polar solvents,and combinations and modifications thereof. In another aspect theextraction solvent is a 1:1 mixture of chloroform and methanol,comprising about 2% acetic acid.

The present invention in one embodiment describes a fingerprint liftmethod for detecting trace amounts of one or more analytes from a solidsubstrate comprising the steps of: providing a cast, a mould or anyother solid impression of a human finger, wherein the cast or the mouldcomprises one or more ridges duplicating the ridges found on the humanfinger, saturating the cast, the mould or the solid impression with anoil, a grease or a lipid by a spraying, a dipping or a coating process,lifting the analytes from the solid substrate by pressing or contactingthe cast, the mould or the solid impression with the substrate,transferring the lifted analytes from the solid substrate to amicroscopic slide or any other suitable solid support, and detecting theone or more analytes by generating a mass spectrum in a massspectrometer, wherein the detection is done by identifying a m/z ratioof the analytes in the mass spectrum.

In one aspect the one or more analytes comprise explosives, drugs, andnarcotics. In another aspect the analyte is cocaine. In another aspectthe analyte is nitroglycerine (NG) and dinitrotoluene (DNT). It will beunderstood by a person skilled in the art that the technique of thepresent invention can be applied to literally hundreds of energeticmaterials. In yet another aspect the mass spectrometer system isequipped with a nanospray source. The system as described in the methodof the present invention comprises: a) an inverted microscopic stagemounted with or capable of holding the solid support comprising the oneor more analytes, b) an extraction system for an extraction of the oneor more analytes from the support followed by a transfer to a detectionsystem, wherein the detection system is coupled to the extractionsystem, wherein the extraction system comprises: (i) a workstationcomprising a plurality of moveable nanopositioners, wherein thenanopositioners are capable of movement in a X-Y-Z plane, wherein thenanopositioners hold one or more probes, grippers, capillary tips or anysuitable accessory for an extraction and transfer of a liquid phase,(ii) a pressure injector for delivery of a pressurized extractionsolvent through the capillary tip, wherein the capillary tip is placedin close proximity to the support, (iii) a voltage source, (iv) ajoystick or a digital controller for controlling the movement of thenanopositioners, and (v) a mass spectrometer connected to the extractionsystem for receiving the one or more analytes transferred by theextraction system. In another aspect the method further comprises thestep of generating a background mass spectrum comprising any otheranalytes that may be expected to be present, the oil, the grease or thelipids or both, wherein the background spectrum is used to correct forinterferences from the other analytes, the oil, the grease or the lipidsor both.

Yet another embodiment describes a fingerprint lift method for detectingtrace amounts of one or more dissolved analytes from a liquid comprisingthe steps of: (i) evaporating the liquid to obtain a solid residue,wherein the solid residue comprises the one or more analytes to bedetected, (ii) providing a cast, a mould or any other solid impressionof a human finger, wherein the cast or the mould comprises one or moreridges duplicating the ridges found on the human finger, (iii)saturating the cast, the mould or the solid impression with an oil, agrease or a lipid by a spraying, a dipping or a coating process, (iv)lifting the analytes from the solid residue by pressing or contactingthe cast, the mould or the solid impression with the residue, (v)transferring the lifted analytes from the solid residue to a microscopicslide or any other suitable solid support, and (vi) detecting the one ormore analytes by generating a mass spectrum in a mass spectrometer,wherein the detection is done by identifying a m/z ratio of the analytesin the mass spectrum.

In one aspect the one or more analytes comprise explosives, drugs, andnarcotics. In another aspect the analyte is cocaine. In another aspectthe analyte is nitroglycerine (NG) and dinitrotoluene (DNT). It will beunderstood by a person skilled in the art that the technique of thepresent invention can be applied to literally hundreds of energeticmaterials. In yet another aspect the mass spectrometer system isequipped with a nanospray source, wherein the system comprises: (i) aninverted microscopic stage mounted with or capable of holding the solidsupport comprising the one or more analyses, and (ii) an extractionsystem for an extraction of the one or more analytes from the supportfollowed by a transfer to a detection system, wherein the detectionsystem is coupled to the extraction system, wherein the extractionsystem comprises: a workstation comprising a plurality of moveablenanopositioners, wherein the nanopositioners are capable of movement ina X-Y-Z plane, wherein the nanopositioners hold one or more probes,grippers, capillary tips or any suitable accessory for an extraction andtransfer of a liquid phase, a pressure injector for delivery of apressurized extraction solvent through the capillary tip, wherein thecapillary tip is placed in close proximity to the support, a voltagesource, a joystick or a digital controller for controlling the movementof the nanopositioners, and a mass spectrometer connected to theextraction system for receiving the one or more analytes transferred bythe extraction system. In yet another aspect the extraction solventcomprises water, polar organic and inorganic solvents, mixtures of polarand non-polar solvents, and combinations and modifications thereof. Inyet another aspect the method further comprises the step of generating abackground mass spectrum comprising any other analytes that may beexpected to be present, the oil, the grease or the lipids or both,wherein the background spectrum is used to correct for interferencesfrom the other analytes, the oil, the grease or the lipids or both.

In one embodiment the instant invention discloses a method for liftingprints, detecting one or more analytes, drug residues, or contaminantsin a mixture, or any combinations thereof comprising the steps of: (i)placing a substrate or a film on top of and in contact with the print,the one or more analytes, drug residues, or contaminants in a mixture,or any combinations thereof, wherein the substrate or film comprises ametal coated surface and (ii) applying a voltage to the substrate or thefilm, wherein the application of the voltage results in a lifting, aretrieval or an adhesion of the print, the one or more analytes, drugresidues, contaminants in a mixture, or any combinations thereof due toa combination of electrostatic and conductive forces. The method asdescribed hereinabove further comprises the step of detecting the one ormore analytes, the drug residues, contaminants in a mixture, or anycombinations thereof by Raman spectroscopy or by generation of a massspectrum in a mass spectrometer, wherein the detection is done byidentifying a m/z ratio of the analytes in the mass spectrum. In oneaspect the step of detection is performed by Surface Enhanced RamanScattering (SERS). In another aspect the step of detection is performedby a mass spectrometer system equipped with a nanospray source. In yetanother aspect the one or more analytes comprise explosives, drugs,narcotics, or any combinations thereof wherein the drugs are selectedfrom the group consisting of cocaine, amphetamines, codeine,hydrocodone, and crystal meth. In other related aspects the substrate orthe film comprises a polymer, a polyester, or any conductive materialcapable of lifting the one or more analytes from a surface and the filmcoating comprises metals selected from gold, silver, or any combinationsthereof, wherein the metals are deposited by physical vapor deposition.

Another embodiment of the instant invention disclosed herein provides amethod for lifting prints, detecting one or more analytes, drugresidues, or contaminants in a mixture, or any combinations thereofcomprising the steps of: i) placing an uncoated first substrate or afirst film on top of and in contact with the print, the one or moreanalytes, drug residues, contaminants in a mixture, or any combinationsthereof, wherein the analyte, the drug residue, contaminants in themixture, or any combinations thereof adhere to and are collected ontothe surface of the first substrate; ii) placing a coated secondsubstrate or a second film on top of and in contact with the firstsubstrate or first film comprising the collected one or more analytes,drug residues, contaminants in a mixture, or any combinations thereof,wherein the second substrate or the second film is a metal coated film;and iii) applying a voltage to the second substrate or the film, whereinthe application of the voltage results in a lifting, a retrieval or anadhesion of the print, the one or more analytes, drug residues,contaminants in a mixture, or any combinations thereof due to acombination of electrostatic and conductive forces. In one aspect thestep of detecting the one or more analytes, the drug residues,contaminants in a mixture, or any combinations thereof by Ramanspectroscopy or by generation of a mass spectrum in a mass spectrometer,wherein the detection is done by identifying a m/z ratio of the analytesin the mass spectrum. In another aspect the one or more analytescomprise explosives, drugs, narcotics, or any combinations thereofwherein the drugs are selected from the group consisting of cocaine,amphetamines, codeine, hydrocodone, and crystal meth. In yet anotheraspect the substrate or the film comprises a polymer, a polyester, orany conductive material capable of lifting the one or more analytes froma surface. In a related aspect the film coating comprises metalsselected from gold, silver, or any combinations thereof, wherein themetals are deposited by physical vapor deposition. In a specific aspectsecond substrate or film is coated with gold. In other aspects the firstsubstrate or film and the second substrate or film may comprise same ordifferent materials and a thickness of the metal coated films rangesfrom 25 nm-100 nm.

In yet another embodiment the present invention provides a method foridentifying lipid components, determining lipid profiles, detectingrelative amounts of different lipids, or any combinations thereof from alipid storing substrate, body, tissue, cell, organelle, lipid droplets(LDs) or any combinations thereof comprising the steps of: i) providinga sample comprising the one or more lipid containing substrates, bodies,tissues, cells, organelles, LDs, or any combinations thereof; ii)providing an extraction system for an extraction of the one or morelipids from the lipid containing tissue, cell, organelle, lipid droplets(LDs) or any combinations thereof followed by a transfer to a detectionsystem, wherein the detection system may be coupled to the extractionsystem; iii) dissolving the one or more lipids by an injection of theextraction solvent onto the substrate; iv) aspirating the dissolvedlipids by the one or more capillary tips; v) injecting the aspiratedlipids into the mass spectrometer to obtain a mass spectrum; and vi)identifying the one or more lipids by a m/z ratio in the mass spectrum.

The extraction system as described hereinabove comprises:

a) a workstation comprising a plurality of moveable nanopositioners,wherein the nanopositioners are capable of movement in a X-Y-Z plane,wherein the nanopositioners hold one or more probes, grippers, capillarytips or any other suitable accessory for an extraction and transfer of aliquid phase;b) a pressure injector for a delivery of a pressurized extractionsolvent through the capillary tip, wherein the capillary tip is placedin close proximity to the lipid containing substrates, bodies, tissues,cells, organelles, LDs, or any combinations thereof;c) a voltage source;d) a joystick or a digital controller for controlling the movement ofthe nanopositioners; ande) a mass spectrometer that is off line or is connected to theextraction system for receiving the one or more dissolved analytestransferred by the extraction system.

The method as described hereinabove further comprises the steps of: i)determining a lipid profile from an extract of lipids or one or morelipid standards; and ii) comparing the identified lipids from the samplewith the lipid profile from the extract of lipids or the lipid standardsto determine the lipid profile of the sample, the relative amounts ofthe lipids in the sample, or any combinations thereof. In a specificaspect the mass spectrometer is equipped with a nanospray source and thesubstrate is mounted on an inverted microscopic stage. In another aspectthe extraction solvent comprises water, polar organic and inorganicsolvents, mixtures of polar and non-polar solvents, and combinations andmodifications thereof. In yet another aspect the extraction solventcomprises 10 mM ammonium acetate in chloroform:methanol (1:1, v/v). Inanother aspect the lipids comprise triacylglycerol (TAG),monoglycerides, diglycerides, triglycerides, lipid derivatives likefatty acid methyl esters (FAME), or any combinations thereof. In anotheraspect the method described herein further comprises the optional stepof isolating and purifying the lipid containing substrate, body, tissue,cell, organelle, LDs, or any combinations thereof.

In one embodiment the present invention relates to a method foridentifying lipids, determining lipid content, profile, or both or anycombinations thereof in a single-lipid droplet (LD) or directly from alipid containing organelle comprising the steps of: i) providing one ormore isolated and purified LDs or lipid containing organelle, whereinthe LDs or the lipid containing organelle are obtained from a plant oran animal source; ii) providing an extraction system for an extractionof the LDs or the lipid containing organelle, followed by a transfer toa detection system, wherein the detection system may be coupled to theextraction system, wherein the extraction system comprises: a) aworkstation comprising a plurality of moveable nanopositioners, whereinthe nanopositioners are capable of movement in a X-Y-Z plane, whereinthe nanopositioners hold one or more probes, grippers, capillary tips orany other suitable accessory for an extraction and transfer of a liquidphase; b) a pressure injector for a delivery of a pressurized extractionsolvent through the capillary tip, wherein the capillary tip is placedin close proximity to the LDs or the lipid containing organelle, whereinthe extraction solvent comprises 10 mM ammonium acetate inchloroform:methanol (1:1, v/v) spiked with a Tri 15:0 triacylglycerol(TAG) standard; c) a voltage source; d) a joystick or a digitalcontroller for controlling the movement of the nanopositioners; and e) amass spectrometer that is off line or is connected to the extractionsystem for receiving the one or more LDs or the lipid containingorganelle transferred by the extraction system; iii) aspirating the LDsor the lipid containing organelle in the extraction solvent by the oneor more capillary tips; iv) injecting the aspirated LDs or the lipidcontaining organelle into the mass spectrometer to obtain a massspectrum; and v) identifying the one or more lipids by a m/z ratio inthe mass spectrum.

The method of the present invention further comprises the steps of:determining a lipid profile from an extract of lipids or one or morelipid standards and comparing the lipids from the LDs or the lipidcontaining organelle with the lipid profile from the extract of lipidsor the lipid standards to determine the lipid profile of the sample, therelative amounts of the lipids in the sample, or any combinationsthereof. In one aspect the mass spectrometer is equipped with ananospray source. In another aspect the detection system may comprise aRaman spectrometer. In yet another aspect the LDs or the lipidcontaining organelles may be stained using a lipid based dye or animaging agent.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1A is a schematic of the nanomanipulator workstation. Twonanopositioners are capable of holding capillary tips used for nanosprayionization. The remaining two positioners utilize end-effectors (eithertungsten probes or microgrippers) for sample manipulation;

FIG. 1B is a schematic of a nanopositoner holding a capillary tip fornanospray ionization. Here the extraction solvent is shown on the rayonfiber. A time-resolved schematic shows the retrieval of analyteparticles into the tip;

FIG. 2A shows the nanomanipulator positioner with the nanospray tipprobing an analyte;

FIG. 2B shows the nanospray ionization source showing the nanospray tipthat was transferred directly from the nanomanipulator;

FIG. 3 shows a rayon fiber doped with analyte before extraction, thecaffeine particle is on the rayon fiber and the nanospray tip is landedin close proximity;

FIG. 4 shows a rayon fiber doped with analyte before extraction with atwo capillary method with a histidine particle between the capillary tipon the left and the nanospray tip on the right;

FIG. 5A is the mass spectrum of cocaine (MW: 303.35 g/mol) taken inpositive ion mode after extraction from a single rayon fiber. The MH⁺peak appears at m/z 304.35. The inset shows the mass spectrum collectedfrom an extraction on the fiber with no analyte present;

FIG. 5B is a blow-up of mass spectrum shown in FIG. 5A between m/z250-350;

FIGS. 6A-6C demonstrate the accumulation of BODIPY in the nanospraycapillary extracted with chloroform while the aqueous solutionsimultaneously fades at: (FIG. 6A)˜0 minutes, (FIG. 6B)˜10 minutes,(FIG. 6C)˜25 minutes;

FIG. 6D shows another uncoated, empty nanospray capillary within thesample confirming the results were not resulting from autofluorescenceof the extraction solvent. Scale Bars ˜100 μm for FIG. 6A and ˜5 μm forFIG. 6D;

FIG. 6E is a nanospray mass spectra of BODIPY 493/503 dye sampledthrough LPME-NMS with 1:1 (v/v) chloroform:methanol containing 2%glacial acetic acid. A dominant peak of 263.3 m/z confirmed that the[BODIPY+H]+ was successfully extracted;

FIG. 7 shows two series of polyethylene glycol (PEG) chain polymers of44 m/z units apart, average masses of 830.5 m/z and 1464.1 m/z, wereextracted with 1:1 (v/v) chloroform:methanol containing 2% glacialacetic acid from vertebrate serum sample that had been processed byaffinity column chromatography;

FIG. 8A shows the acid-catalyzed hydrolysis of trioleate with a [H]+adduct at 885.5 m/z, [NH₄]+ adduct at 903.0 m/z, and [Na]+ adduct at908.0 m/z by LPME-NMS with 1:1 (v/v);

FIG. 8B shows that the diacylglycerol fragment was effectively minimizedby substituting 10 mM ammonium acetate in place of the glacial aceticacid. MS/MS (inset) of trioleate confirmed the characteristicdiacylglycerol fragment at 603.5 m/z;

FIG. 8C is a representative acid-catalyzed hydrolysis spectra by LPMEextraction with 1:1 (v/v) chloroform:methanol containing 2% glacialacetic acid of refined cottonseed oil (black lines) produced anabundance of diacylglycerol fragments (hydrogen adducts) for PL at 575.5m/z, PO at 577.5 m/z, LL at 599.5 m/z, OL at 601.5 m/z, and OO at 603.5m/z where P is Palmitate, L is Linoleate, and O is Oleate. (grey lines)The diacylglycerol fragments were effectively minimized by substituting10 mM ammonium acetate in place of glacial acetic acid which producedtriacylglycerols of primarily ammonium adducts;

FIGS. 9A and 9B show the mass spectra of caffeine and histidinerespectively;

FIG. 10 shows a gunshot residue sample and accompanying mass spectrum;

FIGS. 11A and 11B show ink extraction from a written document with thenanomanipulator;

FIGS. 12A and 12B show a caffeine/soil lift done with the electrostaticlifter;

FIGS. 13A-13D show: (FIG. 13A) caffeine extraction with nanomanipulator,(FIG. 13B) NSI-MS spectrum of caffeine. MH⁺ peak is seen at m/z 195.00,(FIG. 13C) ESI-MS spectrum of cocaine wash from electrostatic lift. MH⁺peak is seen at m/z 304.73, (FIG. 13D) NSI-MS spectrum of cocaineextraction from electrostatic lift. MH⁺ peak is seen at m/z 304.13;

FIG. 14 Bright field image of purified lipid droplets from cotton seed(Gossypium hirsutum) being directly sampled with a nanospray emitter.Scale Bar=10 microns;

FIG. 15A is a mass spectrum of trioleate(1,2,3-tri-(9Z-octadecenoyl)-sn-glycerol) standard dissolved in 1:1CHCl₃/CH₃OH plus 2% acetic acid;

FIG. 15B is a mass spectrum of triacylglycerols extracted directly fromlipid droplets of cotton seed (Gossypium hirsutum) with chemicalstructures of corresponding m/z peaks;

FIG. 16 shows a NSI tip landed near peptide coated solid support;

FIG. 17A is a mass spectrum of a polyglycine peptide sequence bulksample as analyzed by NSI-MS;

FIG. 17B is a mass spectrum of a polyglycine peptide sequence singlebead as analyzed by NSI-MS;

FIG. 18A is an ESI MS/MS spectrum of H-PWSG-NH₂;

FIG. 18B is a NSI MS/MS spectrum of H-PWSG-NH₂;

FIG. 19 shows a microscopic friction ridge detail captured up close;

FIG. 20 shows a fingerprint lift of cocaine/red fluorescent powder;

FIG. 21 shows cocaine extraction from fingerprint lift;

FIG. 22 is a NSI-MS Cocaine Extraction Spectrum. Parent ion peak is atm/z 304.27;

FIGS. 23A and 23B are schematic diagrams of a two-step lifting processwhere, FIG. 23A is a primary lift containing both analyte (white) andmatrix (gray) particles is covered with a gold-coated film and FIG. 23Bis a secondary lift is performed to collect the analyte particles forSERS analysis, leaving the primary lift intact;

FIG. 24 is a Raman spectrum for the caffeine standard on a) Mylar® film,b) 25 nm gold film, c) 60 nm gold film and d) 100 nm gold film;

FIG. 25 is a comparison of the full spectra for a) caffeine on Mylar®and b) caffeine on the 100 nm gold film;

FIG. 26 is a Raman spectrum for cocaine on a) Mylar® film, b) 25 nm goldfilm, c) 60 nm gold film and d) 100 nm gold film;

FIG. 27 is a Raman spectrum for crystal meth on a) Mylar® film, b) 25 nmgold film, c) 60 nm gold film and d) 100 nm gold film;

FIG. 28 is a Raman spectrum for MDMA on a) Mylar® film, b) 25 nm goldfilm, c) 60 nm gold film and d) 100 nm gold film;

FIG. 29 is a comparison of the signal-to-noise ratios for the foursamples on each of the films;

FIGS. 30A-30C are microscope images of the area mapped in the Ramananalysis of cocaine and sand (FIG. 30B), contour map overlaid on theimage (FIG. 30A) showing areas of cocaine signal in grey and white (thethreshold peak intensity for white is 12.23 a.u.), and three-dimensionalplot of the Raman image with the microscope image overlaid (FIG. 30C);

FIGS. 31A and 31B show a schematic of direct organelle massspectrometry. A schematic representation of DOMS using the L200nanomanipulator coupled to nanospray mass spectrometry. (FIG. 31A) thenanopositioner, set on an inverted microscope stage, electronicallycontrols the x, y, and z positioning of a nanospray emitter through auser-operated joystick. The nanospray emitter is prefilled with amicroextraction solvent and is directly connected to a pressureinjector. Approaching purified LDs in buffer, the emitters canselectively load LDs of interest, microextract their lipid contents, andanalyze their compositions by nanospray mass spectrometry, (FIG. 31B)Scale bar represents 2 μm;

FIGS. 32A-32C shows the TAG profiles of purified cottonseed lipiddroplets analyzed by DOMS. (FIG. 32A) representative positive ion, highresolution TAG profile of purified LDs from mature cotton embryos (cv.Coker 312). Dominant TAG species are identified as ammonium adducts[M+NH4]⁺ with peaks labeled according to the three fatty acids presentin each TAG molecular species as identified through subsequent tandem MSanalysis by collision induced dissociation (CID), (FIG. 32B) schematicof tandem MS of a TAG with acyl chains R1, R2, R3. Fragmentation of theR1 ester bond (blue line) produces a corresponding DAG plus hydrogenadduct (blue, R2 and R3) and free fatty acid (FFA) plus ammonia (blue,R1), (FIG. 32C) confirmation of acyl chain composition by tandem MSdetects the diagnostic DAG product ions minus a free fatty acid comparedwith known masses. [94] DAGs represented in multiple TAGs shown are onlylabeled once for clarity. sn-positioning of each acyl chain was notdetermined. P, 16:0-palmitic acid; O, 18:1-oleic acid; L, 18:2-linoleicacid;

FIGS. 33A-33G show the characterization of lipid droplets from higholeic cottonseed mutants. Representative in situ confocal images ofmature cotton embryos from wild type (cv. Coker 312) (FIG. 33A) and atransgenic line expressing a Brassica nonfunctional allele of a delta-12fatty acid desaturase (Bnfad2) stained with BODIPY 493/503, (FIG. 33B).Purified LDs of Coker 312 (FIGS. 33C and 33E) and Bnfad2 (FIGS. 33D and33F) mature embryos visualized in bright field and stained with BODIPY493/503. (FIG. 33G) representative TAG profiles of Bnfad2 (blue) andCoker 312 (red) using DOMS of the purified LDs shown in FIGS. 33C and33D. Scale bars represent 10 μm (FIGS. 33A and 33B) and 2 μm (FIGS.33C-33F). P, 16:0-palmitic acid; O, 18:1-oleic acid; L, 18:2-linoleicacid;

FIGS. 34A-34D show the validation of DOMS with conventional total lipidextracts. Representative TAG profiles from total lipid extracts ofpurified LDs from mature cotton embryos of Coker 312 wild type (FIG.34A) and Bnfad2 transgenic (FIG. 34B) lines. Dominant TAG species areidentified as ammonium adducts [M+NH4]⁺ with peaks labeled according tothe total number of carbons followed by the total number of double bondsfor that particular TAG mass-to charge ratio. Quantitative comparison ofmolecular TAG content (FIG. 34C, Coker 312; FIG. 34D, Bnfad2) acquiredthrough direct organelle mass spectrometry of small LD populations(10-25 LDs) relative to conventional total lipid extracts. Predominantacyl chain combinations were quantified by integrating the absoluteintensities of peaks identified through tandem MS, correcting forisotopic overlap, and converting to molecular percentages. P,16:0-palmitic acid; O, 18:1-oleic acid; L, 18:2-linoleic acid;

FIGS. 35A-35C show single lipid droplet mass spectrometry and lipiddroplet heterogeneity. (FIG. 35A) bright field snapshot image of ananospray emitter directly sampling a single LD. Scale bar represents 5μm, (FIG. 35B) representative TAG profiles with acyl chain designationsof single LD from Tables 2 and 3 (Coker 312 LD6 and Bnfad2 LD6. LD-TAGpeaks are attenuated according to the right axis to show resolution ofpeaks relative to spiked Tri 15:0 TAG standard represented by the leftaxis, (FIG. 35C) heterogeneity of TAG content within single seed LDs(n_(—)7 for both Coker 312 and Bnfad2). Numerical values are in Tables 2and 3. P, 16:0-palmitic acid; O, 18:1-oleic acid; L, 18:2-linoleic acid;

FIGS. 36A-36C show the detection of lipid droplets with cyclic fattyacyl chains in cotton roots. (FIG. 36A) representative confocal image ofBODIPY 493/503 stained LDs of 48-h germinated roots, (FIG. 36B) brightfield image of purified root LDs and resulting TAG profile (FIG. 36C,blue) relative to cotyledon cottonseed LDs (FIG. 36C, red). Cyclic fattyacid abbreviations are as follows: Sc, sterculic acid; Dsc,dihydrosterulic acid. Scale bars represent 10 μm. P, 16:0-palmitic acid;O, 18:1-oleic acid; L, 18:2-linoleic acid;

FIGS. 37A-37C show tandem MS analysis of lipid extracts of purifiedcotton root lipid droplets. In addition to tandem MS of LDs sampledthrough DOMS, precursor-product scans of lipid extracts of purified LDsof cotton roots aided in identification of cyclic fatty acids. Shown areprecursors TAG profiles of malvalic/linoleic. (FIG. 37A) sterculic,(FIG. 37B) dihydrosterculic, (FIG. 37C) fatty acids analyzed in neutralloss mode on a triple quadrupole MS. Dominant TAG species are identifiedas ammonium adducts [M+NH4]⁺ with peaks labeled according to the totalnumber of carbons followed by the total number of double bonds for thatparticular TAG mass-to-charge ratio. Underlined species in themalvalic/linoleic scan (FIG. 37A) correspond to TAGs with at least onesterculic or dihydrosterculic acyl chain; and

FIGS. 38A-38F show the characterization of Arabidopsis leaf and seedlipid droplets. Representative TAG profiles of LDs purified from (FIG.38A) Arabidopsis mature seeds and (FIG. 38B) rosette leaves of40-day-old plants. Labels show the total number of carbons followed bythe total number of double bonds for that particular TAG mass-to-chargeratio. Seed LDs showed an abundance of 20:1/eicosenoic fatty acids,whereas leaf LDs showed typical 18:3/16:3 fatty acids. Representative insitu confocal image, (FIG. 38C) of wild type Arabidopsis mesophylltissue stained with BODIPY 493/503 (red autofluorescence representschloroplasts) and bright field snapshot image, (FIG. 38E) of purifiedArabidopsis leaf LDs. Representative in situ confocal image (FIG. 38D)of wild type Arabidopsis seed LDs and (FIG. 38F) epifluorescence imageof purified Arabidopsis seed LDs stained with BODIPY 493/503. Scale barsrepresent 20 μm ((FIG. 38C), 10 μm ((FIG. 38C, inset), and 5 μm ((FIGS.38D-38F).

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

A technique involving the combination of nanomanipulation and nanospraymass spectrometry (NMS) for single cell, single organelle, andultra-trace molecular analysis is described herein below.

The advent of small volume analysis with mass spectrometry has openedthe door to the study of micron and submicron analytes, and bringing thesampling directly to the area of interest. The nanomanipulator of theinstant invention is a multistage bioworkstation consisting of afour-positioner system that has been directly coupled with nanospraymass spectrometry (NMS). This coupling allows for new significantapplications developments in the areas of trace and document analysiswithin the forensic field. This technique has been applied to tracefiber analysis and electrostatic lifts for illicit drugs, as well asdocument and painting analysis (as seen in FIGS. 11A and 11B). In FIG.11A The document or painting (1104) is placed on the stage of themicroscope. The nanomanipulater assembly (1106) is placed on top of thedocument, underneath the microscope objective (1102). As seen in FIG.11B the ability to connect two of the nanopositioners (1106 and 1108) tothe pressure injector (1114 and 1116) allows for extractions using twocapillary tips. In this case of the tips in lowered towards the document(1104) in order to perform an extraction of the ink. The othernanopositioners (1110 and 1112) can be equipped with tweezers or probesto carry out other processes.

The low detection limits and sample volumes make nanosprayionization-mass spectrometry the ideal instrument for trace analysis.The present inventors have demonstrated the technique by dissolving anelectrostatic particle of cocaine from a fiber and lift, collecting theanalyte solution in a nanospray tip, and transferring the tip directlyto the mass spectrometer to complete the analysis. The technique of thepresent application was applied to document analysis. The importance ofleaving minimal “footprints” behind to retain the integrity of thedocument is one of the most advantageous benefits of this technique.Inks, as well as, paints and pigments were analyzed, illustrating theminimal damage to the document during analysis. The utility of thistechnique is evident through the minimal sample preparation and shortanalysis time. The technique presented herein could improve on currenttrace particulate analysis and document analysis by reducing bothdetection limits and sample size required to complete analysis.

The few applications described hereinabove are only a few among all ofthe possible applications especially with the ability to design andfabricate new end-effectors with unique abilities. The instant inventioncan be applied to direct cellular probing including toxicology studiesand organelle analysis of single cells and also in forensic sciences(e.g., gunshot residue sampled from fibers 1004 as seen in FIG. 10).

The multistage bioworkstation used in the instant invention consists ofa nanomanipulator stage with four nano-positioners which are attached toa cabinet with a piezo voltage source and a pressure injector. Thebioworkstation is mounted to the stage of the inverted microscope; itcan be easily transferred to other microscopes enabling access todifferent types of visualization (confocal vs. wide-field). Theworkstation can hold up to eight nano-positioners allowing multipleprobing tips and end-effectors to be used if needed. There are twocoarse mode nano-positioners which are driven by a stick slip drive. Ithas a PWM signal that is applied to an arm with a ceramic bead attachedto the end. The arms path is an oval; the band sticks to a ceramic platethen slips back which repeats the cycle to generate the movement. Therange of motion is 12 mm in the X and Z axes and 28 mm in the Y axiswith a resolution of 100 nm. There are two fine nano-positioners havingcoarse mode abilities as well as an optional fine mode that has a 10 nmresolution. The fine mode nano-positioners are driven by apiezo-electric crystal, when high voltage is applied to the crystal itexpands. The geometry and orientation of the crystal is responsible forthe range of motion in each axis (X,Y,Z). The range of motion of thefine mode is 100 μm in the Z and X axes and 10 μm in the Y axis. Thepresent inventors have an added motion in being able to manually tiltthe nano-positioners. In total there are 4 degrees of motion.

The bio-workstation was coupled to nanospray mass spectrometry as shownin the image 100 in FIG. 1A. The nanomanipulator is placed on top of themicroscope stage (114) on an inverted microscope (116). Thenanomanipulator consists of four positioners; two which can be affixedwith capillaries for nanospray extraction (102 and 108) the other twopositioners can be used for probes and grippers (104 and 106). Thespecimen to be manipulated; for example, analyte particles (112) orfibers (110) are placed underneath the positioners (102, 104, 106, and108). This coupling was made possible by adjusting the coarsenano-positioner to hold a nanospray tip. The process includes filling ananospray tip with a nanospray solvent and an adducting ion theninserting it into the nanospray source and breaking the tip open. Thenanospray tip is then transferred to the nano-positioner and the tiplanded near the object of interest. The pressure injector gives acontrolled amount of pressure (0.001 psi to 60 psi) to the tip to injectthe solvent and dissolves the analyte of interest followed by pullingthe dissolved analyte back into the tip. The nanospray tip is thentransferred to the nanospray source and the analyte is analyzed based onits m/z as well as its fragment pattern, MS″, this coupling allows forthe analysis of multiple compounds.

The bio-workstation can also be coupled to other instruments includingmicrofluidic systems allowing for the separation and analysis of amixture of compounds. Microfluidic systems are now being directlycoupled to mass spectrometry to analyze samples allowing for futuresampling, separation, and analysis.

The capillary tips (100 nm to 5 μM diameters) are attached to thenano-positioners which are connected to a pressure injector with aninjection pressure of 60 psi and a fill pressure of 24 mmHg. Capillarytips can be used to sample directly from cells. The present inventorshave set up sequences with up to four capillaries in order to increaseproductivity and expand the types of studies that can be performed. Thepressure injector has a hold function allowing a beveled capillary tipto hold a cell if necessary. Capillary tips are as small as 1 μm withusing a pressure of 60 psi to overcome the surface tension of water. Ittakes less to overcome the surface tension of different solvents withlower viscosity. Surfactants may be used to lower the surface tensionand allow the injection and fill to take place.

In the past micromanipulation can be accomplished with fine motordrives. The instant invention however, includes the fine stick-shifttechnology of piezio allowing true nano manipulation offering a verydistinct advantage. The stick-shift motion is from 3 nm to 100 nm. Inaddition, current manipulation techniques have a resolution of only 1 μmmotion, ultra fine analysis requires better resolution, as provided bythe present invention.

Direct probing from a sample surface directly coupled to massspectrometry (MS) is a useful tool, helping to eliminate samplepreparation and analysis time. Currently, there are three techniques atthe forefront of direct-coupled surface sampling MS: desorptionelectrospray ionization (DESI) [1], surface sampling probe electrosprayionization [2], and dielectric barrier discharge ionization source(DBDI) [3]. DESI sprays charged solvent droplets onto an ambient surfacewhich ionizes neutral analytes. The analytes are then desorbed from thesurface and analyzed using MS [1,4]. DESI has been used to detect traceamounts of explosives as well as sampling directly from human skin[5,6]. Surface sampling probe electrospray ionization uses a liquidjunction between the electrospray source and the surface to dissolve andthen ionize the analyte, which is then electrosprayed into the MS [2,7].This method has been used to sample drugs directly from thin tissueslices [8]. DBDI uses a dielectric barrier discharge to create a stableplasma flow that desorbs and ionizes the sample off of an ambientsurface, then analyzes it using MS [3]. All of these techniques havegreat utility, but need a relatively large area (20-100 μm²) foranalysis.

Micromanipulation is a significant tool in the biological and chemicalsciences. It is utilized primarily to manipulate small particles andcellular materials because of its precise movements. It is currentlybeing used in the biological sciences for single cell transfer [9], toisolate specific bacterial cells from a group [10], and it has also beenemployed for sample preparation for MS analysis [11]. Mitochondria havebeen extracted from cells using micromanipulation and subsequentlyanalyzed using electrophoresis [12].

Nanomanipulation as discussed herein will generally refer to the use ofa commercially available instrument from Zyvex (Richardson, Tex.) thathas the capability of manipulating samples as well as extracting targetanalytes from those samples. As the nanomanipulator was designed to beutilized with electron microscopy, the manipulator end-effectors havebetter than 5 nm translational resolution, which is beyond the opticallimit. This allows for new advances in the biological and chemicalsciences to be made through precise movements and minimally invasivesample manipulation.

Example I Trace Analyte Sampling

One of the current methods of probing trace analytes is the swab method,whereby an object's surface is swabbed using a textile sampling swabthat is then put into a solvent to extract the analyte of interest [13].This method is not the best way to collect trace analytes because ofanalyte losses and dilution of analyte concentration. Repetitivehandling of the analyte, associated with multistep processes, can leadto sample contamination [14]. Additional difficulties arise whenattempting to swab a single fiber, as analyte concentrations will bevery low, making analysis difficult. Improvement in trace analytesampling is needed to more accurately solve problems and collect traceevidence.

Mass spectrometry is a useful tool for trace analysis because of itshigh sensitivity, allowing it to be useful for a wide variety ofcompounds. Nanospray is an ideal ionization source to couple tonanomanipulation, because it reduces sample preparation time andrequires a small concentration of analyte (pmol/μL). Nanospray is anionization technique that, at best, requires 300 attograms (10⁻¹⁸ g) ofanalyte with a minimum volume of 300 nL. Additionally, it is not asaffected by salts as electrospray ionization, which further reducessample preparation [15]. Liquid chromatography-electrosprayionization-mass spectrometry (LC-ESI-MS) has been used in the analysisof explosives [16] and other trace analytes because of the ability todeconvolute a large sample matrix. Using nanomanipulation, the nanospraytip is brought to the analyte to discriminate particle selection, whichwould help to both deconvolute the spectra and elucidate the identity ofthe analyte. These techniques can be applied to trace analysis asdescribed herein, expanding current abilities, so that the trace is nowable to be extracted and analyzed.

Materials: The solvents and chemicals utilized were chloroform (CHCl₃),glacial acetic acid (HOAc), Optima* LC/MS methanol (MeOH), and caffeine(Thermo Fisher Scientific Inc., Waltham, Mass.); no further purificationwas necessary. A sample of freebase cocaine was provided by theUniversity of North Texas Police Department (Denton, Tex.). Milliporewater was obtained using the Milli-QUF Plus (Millipore, Billerica,Mass.) with better than 18 MX salt content. Glass-bottom dishes wereused to hold our samples (Mat Tek Corp., Ashland, Mass.), and analyteswere probed from 100% rayon white bemberg lining. The MS utilized was anLCQ DECA XP Plus (Thermo Finnigan, San Jose, Calif.) with a nanosprayionization source (Proxeon Biosystems, Odense, Denmark). The inventorshave also attached the nanospray source onto a a Thermo Finnigan TSQ7000 Triple Quadrupole to run a similar analysis. An L200nanomanipulator (Zyvex, Richardson, Tex.), coupled to a TE2000UMicroscope (Nikon, Melville, N.J.) and a PE2000b four-channel pressureinjector (MicroData Instrument Inc., S. Plainfield, N.J.) were used toretrieve the analyte from the fiber.

Methods: The L-200 nanomanipulator 100 is mounted to a Nikon TE2000Uinverted microscope (116) and microscope stage (114). Thenanomanipulator employs four nanopositioners (102, 104, 106, and 108)that can be controlled using a joystick and/or digital input (FIG. 1A).The nanopositioners have two modes of action that allow for precisecontrol of their movements. In the coarse mode of action, thenanopositioners have a range of motion of 12 mm in the X and Z axes and28 mm in the Y axis. The fine mode of action allows for a range ofmotion of 100 μm in the X and Z axes and 10 μm in the Y axis. Thenanopositioners are further distinguished by the type of manipulationtools they use. Two nanopositioners are capable of holding end-effectors(either tungsten probes 106 or microgrippers 104) that can be utilizedin either the coarse or fine mode with 3.4 nm translational resolution.The remaining two nanopositioners (102 and 108), which are run in coarsemode only, hold capillary tips and are capable of 100 nm translationalresolution (FIG. 1B). As seen in FIG. 1B the nanopositioner (152) isconnected to a pressure injector (156) via plastic tubing (158). Thepositioner is controlled in the x, y, and z directions (154) and placedin close proximity to the fiber (164) which is on the microscope stage(166). The nanospray tip (160) is preloaded with extraction solvent(162). The solvent is injected onto the sample (168). The PE2000bpressure injector (156) is used to supply up to 60 psi of injectionpressure and 24 inches Hg of fill vacuum to the capillaries, allowing usto retrieve the analyte of interest. The capability of thenanomanipulator to hold up to eight nanopositioners is beneficialbecause it allows one to conduct multiple probes simultaneously andthus, increases the instrument's capabilities and efficiency.

The Au/Pd-plated nanospray tips (160) were loaded with an appropriatesolvent, and then the tip was broken using the nanospray source head. Ablank was run to determine any solvent contamination, and a backgroundspectrum of the solvent was taken. The tip was then transferred to thenanomanipulator for trace analyte probing from a rayon fiber (164) thatwas doped with the analyte of interest and placed in a glass-bottomdish. The rayon fiber (164) was tacked down to minimize the movement ofthe fiber and, therefore, the movement of the analyte on the fiber. Theparticle of interest was found on the fiber, and then the nanospray tip(160) was landed near it, <1 μm away. The nanospray tip then injectedthe solvent (162) onto the analyte. After the analyte had dissolved, thesolvent/analyte solution was retrieved back into the tip (160). Thenanospray tip (160) was then transferred directly to the nanosprayionization source and the sample analyzed.

FIG. 2A shows one of the positioners (208) of the nanomanipulator with ananospray tip (202) retrieving an analyte. The sample of interest isplaced in a dish (204) which is placed on the microscope stage (210).The nanopositioner (208) is affixed with a nanospray capillary (202) andplaced in close proximity to the sample. A pressure injector isconnected to the positioner with plastic tubing (206). FIG. 2B shows thetip mounted onto the nanospray ionization source (258). Cocaine was usedto illustrate this technique. The nanospray capillary (252) is placed inthe nanospray housing (258). A mini light (260) is attached to thecamera mount (204) is shone on the mass spectrometer inlet (254 and256). When sampling, 50:50 MeOH:H₂O with 1% HOAc was used as the solventand 3 μL was loaded into the nanospray tip. The tip (406) was landednext to the analyte (404) as shown in FIG. 3. A particle (404) isadhered to a rayon fiber (402) and a nanospray capillary (406) is placednear the particle for extraction. The nanomanipulator used an injectionpressure of 20.8 psi for a duration of 11 ms delivered from the pressureinjector and a fill pressure of 65.0 psi with a fill time of 50 ms. Thesample was then analyzed in the positive ion mode using a 2 kVextraction voltage on the NSI-MS. The mass spectrum of cocaine can beseen in FIGS. 5A and 5B.

Cocaine trace particles were sampled directly from a single rayon fiberusing the nanomanipulator and then analyzed using NSI-MS. FIG. 5A showsthe mass spectrum of cocaine after directly probing a trace particlefrom the rayon fiber. As analysis was completed by NSI, the MH⁺ peak ismost prominent and appears at m/z 304.35, as well as the characteristicfragment peak at m/z 181.94. The inset of FIG. 5A displays the blankwhen the solvent was allowed to extract on the fiber with no analytepresent. As can be seen from the inset, the fiber contributed noappreciable peaks to the mass spectrum of cocaine. FIG. 5B displays ablowup of the m/z 250-350 range.

The results clearly show that the nanomanipulator coupled to NSI-MS isan effective instrument to probe trace analytes from fibers. It is animprovement in trace analyte probing from a single fiber, allowing fornew experimental procedures to be created and smaller amounts of analyteto be sampled. The nanomanipulator reduces cost of sampling from fibersbecause of the minimal sample preparation and the reduced analysis time.Computer encoding of the positioners may be implemented to automate theprocedure. Being able to recover trace analytes from a single fiberallows for better analysis of crime scenes, and the reduced sample sizeand volume required for NSI-MS allows for the ability to retrieve ahigher sample concentration. Although the research for this article hasfocused on cocaine, this technique has also been applied to otherparticulate analyte standards including caffeine and histidine (FIGS. 9Aand 9B), and the limit of detection by NSI-MS is on the order of 7 pg (1pg=10⁻¹² g) for histidine, which demonstrates the extreme sensitivityachievable by this technique. This technique has also been applied toanalysis of single organelles and protein extraction from silicon beads,currently being developed by the present inventors. It is important tohave a solvent to dissolve the sample as well as provide a steady sprayflow. Some nonpolar compounds will not dissolve in any of the solventsappropriate for nanospray, so it is important to utilize a two capillarysystem with one capillary containing a solvent to dissolve the analyte,and the other capillary containing the nanospray solvent that retrievesthe dissolved analyte. FIG. 4 illustrates the two capillary system usedwith some nonpolar analytes. A particle (412) is adhered to a rayonfiber (408) and two nanospray capillaries (410 and 414) are placed inclose proximity to the particle for extraction. Diffusion will occur,and the small amount of nonpolar solvent with the analyte of interestwill mix with the nanospray solvent, and the resulting solvent/analytemixture can be analyzed using the NSI-MS. The nanomanipulator is alsocapable of liquid-liquid phase microextractions to sample trace analytesand gain higher sample concentration from a dilute analyte in a liquidsample and then complete analysis.

The example discussed hereinabove centered around the analysis ofparticulates on fibers which are particularly well suited for analysisby an inverted microscope but that by no means limits this method tofibers. The technique presented here works equally well for hair, paper,money, computer chips, treated wood, laminate, metal, manipulatedsurfaces including mylar electrostatic lift films, plastic, or any othersurface where trace particulate analytes are expected. Difficultiesarise with some of these specimens because they are not all equallysuited to examination by an inverted microscope. In this case, thenanomanipulator stage could be transferred to a noninverted microscope.

Example II Liquid-Phase Microextraction-Coupled NSI

Advancements made in chemical separation instrumentation have coincidedwith the development of liquid-phase microextraction (LPME) in whichsmall volumes (nL to μL) are used to partition and concentrate analytesbased on their solubility within two immiscible or partially miscibleliquid phases. Implementation of this miniaturized liquid-liquidextraction (LLE) method has traditionally been carried out as asingle-drop solvent extraction [18, 19] or through a porous polymerichollow fiber [20]. Each method enhances separation and concentrationefficiency for many applications relative to the LLE and allows thecoupling to analytical instrumentation developed for minimal samplevolumes.

The first drop-in-drop solvent extraction method (SDME) as developed byLiu and Dasgupta [21] utilized a multitube assembly to suspend awater-immiscible organic microdrop (˜1.3 μL) within a largercontinuously supplied aqueous droplet. The apparatus measured theconcentration of the analyte through a fiber optic absorbance detectorand was amenable to liquid/gas chromatography by pumping away theconcentrated analyte. Jeannot and Cantwell [22, 23] simultaneouslydeveloped a LMPE apparatus in which a gas chromatographic syringesuspended an organic microdrop that could be withdrawn and directlyinjected in the GC. The more recent development of continuous-flowmicroextraction (CFME) [24] utilized an HPLC solvent delivery systemforcing the organic microdrop to continuously make contact with a freshand flowing sample solution. In addition, a solvent extraction apparatusdeveloped for MALDI experiments [25] forms a nanoliter size organicdroplet surrounded by an aqueous sample within a micropipette tip thatcan subsequently be deposited on a MALDI matrix for direct MS analysis.Alternatively, Shen and Lee [20] developed a hollow-fiber solventextraction method that contains organic solvent within a hollow fibrousmembrane immersed in aqueous solvent amenable to GC.

The improvements in nanospray mass spectrometry (NMS) in terms ofversatility and sensitivity have led to an emphasis on analyzing smallmolecular concentrations and volumes. Nanomanipulation has traditionallybeen discussed in terms of manipulation of nanometer size objects with ananometer size end-effector with (sub)nanometer precision [26]. Thepresent inventors have developed a versatile LPME technique fornanoliter to microliter volumes that is amenable to direct NMS analysis(LPME-NMS). A multistage bioworkstation (FIG. 1A) consisting of fournanopositioners was coupled to an inverted microscope allowing thepossibility to visualize, manipulate and analyze small samples. Theutility of LPME-NMS was illustrated by extracting and analyzingmolecules from a fluorescent dye dissolved in an aqueous solvent,non-polar polymer additives in vertebrate serum, and triacylglycerolswithin industrial refined cottonseed oil.

Sample Preparation: BODIPY (493/503, D-3922),4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene, afluorescent neutral lipophilic dye was purchased from Invitrogen(Carlsbad, Calif.). Tripentadecanoin (1,2,3-Tripentadecanoylglycerol)was purchased from Sigma-Aldrich (St. Louis, Mo.). High purityOptima™-grade solvents were purchased from Fisher Scientific (Hampton,N.H.). Cottonseed oil processing fractions were provided through thecoordination of David Kinard (National Cottonseed Products Association)from a refinery in west Texas. The vertebrate serum sample was preparedby Proteintech Group Inc. (Chicago, Ill.) from an antibody preparationdirected to recombinant plant fatty acid amide hydrolase and processedby affinity column chromatography.

Nanomanipulator: The Biometric L200 nanomanipulator workstationdeveloped by Zyvex (Richardson, Tex.) combines four nanopositioners(FIG. 1A) with a piezo voltage source and a pressure injector situatedon an inverted microscope stage (TE2000U, Nikon, Melville, N.Y.). Thenanomanipulator has two modes of motion along the X, Y, and Z axes. Thefine mode has 100 microns of travel in the X and Z axis and 10 micronsof travel in the Y axis with 3.4 nm resolution controlled bypiezo-electric crystals. The coarse mode has 12 mm of travel in the Xaxis and Z axis and 28 mm of travel in the Y axis with 100 nmresolution. The positioners consist of end-effectors made up of sixisolated, low impedance electrical connections and two glass capillaryattachments. The end-effectors are used for manipulation and, if needed,low impedance electrical characterization. Probes and capillariesattached to the positioners can be manually landed onto the sample andmanipulated electronically using a joystick to control theend-effector's position. The capillaries are connected by Teflon tubingto a PM 2000B Programmable 4-Channel Pressure Injector (MicrodataInstruments, South Plainfield, N.J.).

Liquid-phase microextraction: In LPME a liquid phase (Phase I, aqueousin practice) containing the analyte of interest is in direct contactwith an immiscible extractant liquid phase (Phase II, organic solventsin practice) [27]. The analyte of interest will then diffuse into itspreferred phase based on its distribution coefficient κ [27]. There aremany experimental factors that influence the analyte's extractioncapability including its compatibility within the phases, pH of thephases, extraction time and agitation of phases, as well as the volumeratio of the extractant and donor phases [28]. Thermodynamic and kineticconsiderations must also be taken in account to predict extractionbehavior [22]. The thermodynamic equilibrium concentration in theorganic phase

$\begin{matrix}{C_{o,{eq}} = {{\kappa\; C_{{aq},{eq}}} = \frac{\kappa\; C_{{aq},{initial}}}{1 + {\kappa\;{V_{o}/V_{aq}}}}}} & (1)\end{matrix}$where Caq,initial and Caq,eq are the initial and equilibrium aqueousphase concentrations, demonstrates that although it might not befeasible to reach equilibrium due to time constraints, a sufficientlylarge distribution coefficient, κ, is necessary for efficient extraction[22]. In order to maximize K and concentrate the analyte in theextracted phase, it is crucial to minimize the volume ratio Vo/Vaq ofthe organic and aqueous phases [22]. Kinetically, the observed rateconstant (s−1) validates that for rapid analysis the interfacial area(Ai) and mass transfer parameters (β0) must be maximized and the volumeof the aqueous phase (Vaq) minimized [22].

$\begin{matrix}{k = {\frac{A_{i}}{v_{o}}{{\overset{\_}{\beta}}_{o}( {{\kappa\frac{V_{o}}{V_{aq}}} + 1} )}}} & (2)\end{matrix}$

Liquid-phase microextraction was carried out by back-loading anextraction solvent (typically 3 μL) with GELoader™ tips (Eppendorf,Westbury, N.Y.) into the nanospray capillary (FIG. 1B). The nanospraycapillary mounted to the L200 nanomanipulator was subjected to apositive balance pressure (<1.0 psi) to minimize capillary action at thetip opening while breaking through the sample's surface. A programmed C#script was used to control the agitation of the capillary bytransversing the plated sample parallel to the capillary opening. The C#script has the flexibility to vary the velocity (0 to ˜500 μm/sec),direction (x,y,z) and time to carry out a specific agitation pattern.The program carried out for the LMPE trials typically proceeded for 10minutes back-and-forth parallel to the capillary opening multiple timesat 500 μm/sec while also running perpendicular to the capillary openingat approximately 20 μm/sec to perform LMPE on as much of the sample aspossible. The capillary was removed and mounted to the nanospray sourcefor immediate analysis.

Mass Spectrometry: A Proxeon nanospray source (Proxeon Biosystems,Odense, Denmark) was mounted to a Thermo LCQ Deca XP Plus quadrupole iontrap (Thermo Fisher Scientific Inc., Waltham, Mass.). Samples in 1:1(v/v) chloroform:methanol containing 1-2% glacial acetic acid (oralternatively, 10 mM aqueous ammonium acetate where noted) were infusedthrough New Objective (Woburn, Mass.) Econo12 PicoTip™ Emitter platinumcoated nanospray capillaries (1±0.2 μm). The ion source conditionsconsisted of a 0.6 to 1.2 kV spray voltage, an ion-transfer capillarytemperature of 275° C., and an ion-transfer capillary voltage of 3.0 V.Mass spectra were acquired using the LCQ Tune software program in thepositive ion mode with 3 microscans and a continuous acquisition time.The mass spectra were analyzed with the XCalibur 2.0 software package.Tandem mass spectrometry (MS-MS) performed on the cottonseed oil sampleswas used to confirm the identity of the triacylglycerols (data notshown). Tandem spectra were acquired in positive ion mode with a typicalisolation width of 5.0 m/z, normalized collision energy of 35%,activation Q of 0.250, and an activation time of 30 milliseconds.

The development of LPME-NMS was first visualized by extracting theneutral lipid-specific fluorescent dye, BODIPY 493/503, within anaqueous background (2.5 μg/mL) via an uncoated chloroform-loadedcapillary. The dye quickly concentrated within the pulled capillary tipportion in a time-dependent manner and was effectively extracted fromthe aqueous solution (FIGS. 6A-6C). The intense and prolongedfluorescence within the pulled capillary tip portion suggests amultifold concentration took place within nanoliters of chloroform.Negative control LPME experiments performed with an empty capillary(FIG. 6D, NC tip on the right), an aqueous loaded capillary (not shown),as well as an aqueous solution lacking the BODIPY dye (not shown)confirmed the validity of the LPME technique and the fluorescence withinthe capillary tip was not due to autofluorescence of the extractionsolvent. Bearing in mind that uncoated nanospray capillaries could notbe used directly for MS analysis, efforts were made in subsequentstudies to eliminate the extraneous transfer step of the extract to acoated capillary and perform for the first time a direct coupling ofLPME and NMS. A series of 10 minute BODIPY extractions with coatednanospray capillaries confirmed a dominant BODIPY peak at m/z 263.3(FIG. 6E) supporting compositional consistency within the technique.

The selective extraction of lipophilic compounds from a complexbiological sample was optimized to analyze extracts directly within thesampling capillary as a means to eliminate losses and streamline thedirect coupling of LPME to NMS. Toward this goal, a sample of rabbitserum that had been subjected to affinity column chromatography (topurify antigen-specific antibodies) was sampled by performing theextraction directly with a solution (1:1 (v/v) choloroform:methanol plus2% glacial acetic acid) that served as both an extraction solvent and aninfusion solvent for ionization. The chloroform-methanol mixture is acommon extraction solvent for lipophilic molecules [29] and its ratiocan be adjusted to preferentially select for more or less polaranalytes. However, due to the high volatility, very low surface tension,and low dielectric constant of chloroform [30], it was established theratio has to be carefully considered for direct coupling of LPME to NMS.Other combinations of organic infusion/extraction solvents may beselected for a preferential extraction but it is important to considerwhether the solution will still serve as an appropriate nanosprayionization solvent [30]. NMS confirmed that a polymer series ofpolyethylene glycol (PEG at 44 m/z apart), a common additive tostabilize affinity-purified antibody preparations, was selectivelyextracted from the serum samples (FIG. 7). The abundance of PEG and itshigh affinity for the extraction solvent likely overwhelmed theappearance of other less prominent lipophilic compounds that might havebeen present, such as triacylglycerols and cholesterol, especially sincetheir concentrations would have been extremely low in theseaffinity-fractionated serum samples [31].

Additional studies were designed to profile multiple biomolecules in acomplex mixture of industrial relevance. Trioleate(1,2,3-tri-(9Z-octadecenoyl)-glycerol) was used to standardize LPME-NMSunder ideal extraction and purification conditions. Although trioleatewas not within an aqueous background the compound (liquid at roomtemperature) is slightly immiscible with static organic solvents insidethe pulled tip region of the capillary. Interestingly, upon extractionit was evident that under acidic conditions (2% glacial acetic acid asthe protonation source) an acid-catalyzed hydrolysis reaction (FIG. 15A)[32] of the trioleate was taking place within the capillary tip toproduce the corresponding diacylglycerol fragment, dioleate(1,2-di-(9z-octadecenoyl)-glycerol) (FIG. 8A). It was also apparent thattriacylglycerols had a higher affinity for sodium and ammonium cationsthan for hydrogen ions present in solution (FIG. 15B).

LPME-NMS of industrially refined, bleached and deodorized cottonseed oil(FIG. 8C, black lines) was then analyzed under identical extractionconditions as trioleate to demonstrate the profiling of multipletriacylglycerols within a single medium. FIG. 14 is a bright field imageof purified lipid droplets (1404) from cotton seed (Gossypium hirsulum)being directly sampled with a nanospray emitter (1402). Molecularspecies profiles of triacylglycerols within cottonseed oil wereestablished by a combination of direct infusion of refined cottonseedoil, oil body extracts of cottonseed embryos under the same ionizationconditions (not shown), and previous literature values [33-36]. Underacidic conditions, LPME-NMS of cottonseed oil revealed that theproduction of diacylglycerols was prominent relative to theconcentration of triacylglycerols within a relative short extractionperiod.

The acid-catalyzed hydrolysis reactions were reduced by substituting 10mM ammonium acetate (FIG. 8B and FIG. 8C grey lines) as the adductionsource. The reproducibility of the technique was evaluated in a separateset of experiments by adding tripentadecanoin(1,2,3-tripentadecanoylglycerol) as an internal standard at 1/12 themass of cottonseed oil. Although a solid at room temperature, mildheating at 55° C. of tripentadecanoin within the cottonseed oil producesa homogenous sample before LPME was carried out. Three replicateextractions were carried out for each time point 30, 120, and 240seconds with the first two minute-scan by NMS (Table 1). The first twominutes of the nanospray allowed sufficient time for the elimination ofbackground ions at the longest time-point and was chosen to demonstratethe reproducibility of concentration within the pulled tip region(BODIPY results above show a multifold concentration in the tip). Asexpected the absolute abundance of the internal standard and cottonseedoil increased linearly (R²=0.944) over time with the relativeconcentration of triacylglycerols to internal standard approaching thetheoretical amount (Table 1). Hence the LPME-NMS based approach allowsfor the rapid quantitative analysis of industrially derived, vegetableoil products. Moreover, these studies indicated the expanded utility ofmonitoring controlled chemical reactions within nanospray capillariesduring extraction and direct analysis by NMS.

The variation in extraction efficiency and analyte quantification islikely a consequence of the minimal interfacial interaction, control ofthe balance pressure bringing the two interfaces together, surfacecharge effects and limited agitation at the capillary tip opening.Considering that the interfacial area of an average nano-capillary isfrom ˜0.50 to ˜1.15 μm2 (diameter 0.8 to 1.2 μm), the mass transfer ofthe extracted molecule must be significantly large to concentrate thesolute within the organic phase. The interfacial area is significantlyless than single organic drops with other current methods [36] andpushes the limits for a kinetically favorable transfer event. Theextraction conditions above attempted to take advantage of the highsolubility between lipophilic compounds and organic solvents as well asa concentration gradient at the interfacial barrier. The balancepressure was manually controlled due to the slight variations within tipdiameter. This parameter is the most difficult to control in attemptingto force the two interfaces to make contact without essentiallyinjecting a portion of the organic solvent onto the aqueous sample. Thesurface charge effects are evident with coated nanospray capillaries incomplex solutions or cellular material as “debris” tends to migratetowards and attach itself to the tip openings (even with a positivebalance pressure at the tip end). The agitation of the capillary tip wasrelatively mild compared to vortexing during liquid-liquid extraction ormicrofluidic devices. The velocity with which the capillaries movethrough the sample could likely be increased to simulate a more vigorousextraction. Nonetheless, inclusion of internal standards in the samplesdemonstrates the accurate quantification capabilities of thissmall-volume approach.

TABLE 1 Reproducibility of LMPE-NMS for extracting cottonseed oil.Average RSD of Average Total Predicted Predicted Extraction Time IonCount C/S TAG C/S TAG (seconds) (×10⁷) Quantity (g)^(a) Quantity (%)  30s (n = 3) 1.8 0.113 1.4 120 s (n = 3) 3.7 0.125 2.0 240 s (n = 3) 10.80.142 0.5 C/S—Cottonseed Oil, TAG—Triacylglycerol, RSD—Relative StandardDeviation ^(a)The average predicted cottonseed TAG quantity extractedwas determined through back calculation of absolute peak areas versus aknown quantity of internal standard.

The present inventors have described a novel instrument and a method ofnanomanipulation coupled to NSI-MS as an effective tool to analyze traceanalytes found on fibers, with the successful analysis of cocaine. Theinventors recovered trace particles of cocaine from rayon fibers usingthe nanomanipulator and subsequently analyzed the trace by directlytaking the sample from the nanomanipulator to the NSI-MS (FIGS. 13C and13D). The inventors have also successfully analyzed caffeine using themethod described herein (FIGS. 13A and 13B). In FIG. 13A the particle ofinterest (1306) is combined with a soil mixture (1304) andelectrostatically lifted. The nanospray capillary (1302) is placed nearthe analyte particle for extraction. These examples clearly demonstratethe functionality and utility of the nanomanipulator coupled to NSI-MSto improve upon the current methods of analysis of trace analytes foundon fibers. In the field of trace analysis, preconcentration techniquesproduce only positive results for metal chelates; therefore, a betteroption might be a preconcentration technique like evaporation,extraction methodologies, or changing the sampling procedure to obtain amore concentrated sample [17]. Advantages of NSI-MS include having thecapability to analyze samples with limited volumes (as low as 300 nL)and low limits of detection and would eliminate the need for any suchpreconcentration technique.

Some applications of NSI-MS are shown in FIGS. 16, 17A, 17B, 18A, and18B. FIG. 16 shows a NSI tip (1602) landed near peptide coated solidsupport (1604). FIGS. 17A and 17B show mass spectra of a polyglycinepeptide sequence bulk sample and a polyglycine peptide sequence singlebead analyzed by NSI-MS, respectively. FIGS. 18A and 18B showcomparative mass spectra of H-PWSG-NH2 as analyzed by ESI MS/MS and NSIMS/MS, respectively.

The inventors further describe a LMPE-NMS approach to rapidly samplecomplex mixtures of industrial, chemical, and biological materials forcompositional analysis. The implementation of a few design modificationssuch as improving the effective interfacial area, balance pressure, andagitation of the extraction would result in additional quantitativeability for reproducibility and quality control. Industrially, thetechnique described hereinabove could be an alternative method toquickly sample compounds that are only produced in micro-quantities in aminimal number of steps eliminating sampling methods that traditionallyresult in significant analyte losses. Chemically, the technique could beused to monitor reactions of a compound that became selectivelyextracted upon production. Finally, the L200 nanomanipulator system,with control of multiple end-effectors (FIG. 1A) of the presentinvention also provides the possibility of performing LMPE-NMS withinminiaturized biological systems as a system to profile compounds byselective extraction while visualizing samples.

Example III Retrieval of Drug and Explosive Residues

The retrieval of drug and explosive residues in ultra-trace amountsposes a challenging task for most trace analysts due mostly toinstrumental limitations. The instrumental analysis of ultra-traceamounts serves as a hindrance to obtaining productive results. Detectionof extremely small quantities is usually beyond most operationalstandards of instruments and cannot be done with quality results. Thisissue has prompted the development of techniques combined withinstruments to achieve quality results that include higher sensitivityand selectivity of analytes. However, the retrieval of drug andexplosive residues from fingerprint impressions is a method that doesnot receive much attention. Although there are many techniques that canimage the friction ridge detail of fingerprint impressions, not manyadvance their techniques to the extraction of particulates contained byimpressions. Electrostatic lifting applies the concept of imaging theparticulates of soil and other particles (1206) to outlining thefootwear impressions made on surfaces (FIGS. 12A and 12B). In FIG. 12AThe mylar film (1204) is used to electrostatically lift particles (1206)from a surface (1202) by hand (1208). In FIG. 12B the voltage insulator(1226) houses the probe (1224) used to apply voltage to mylar film(1222) which is placed on top of the surface (1220). The presentinventors have used ruthenium tetroxide (RTX) as a developing techniqueand not just as a powder. Fingerprinting makes use of different types ofpowders specific to developing fingerprints in order to record frictionridge detail. Chemical imaging of trace amounts of residue fromfingerprints as been implemented in the identification of analytesaccording to their chemical signature exhibited in spectra through theuse of infrared spectromicroscopy [37]. However, since most impressionsare made at the scene of the crime, it is not always a simple task toretrieve these particles for analysis. The amount that is there may notbe enough for analysis as well. The modification of GC-MS method isanother technique that has been implemented to facilitate thepreparation of residue components of fingerprints in order identifyanalytes present [38]. This method still requires a certain amount ofpreparation of the components retrieved before instrumental analysis cancommence.

The detection of ultra-trace amounts of explosives is a task that oftenbecomes daunting due to the multiplicity of factors involved in doingexplosives analysis. The chemical makeup of explosives variestremendously making it very difficult to use a standard method ofanalysis [39]. Solvent systems often vary according to type of explosiveand instrumental method utilized. There is a multitude ofinstrumentation designed for explosive residue detection eachimplementing a unique methodology of analysis. Most of these types ofanalysis are elemental based to identify components in the explosivemixtures so as to pinpoint one particular type of explosive. A range oftechnical approaches have been researched and tested toward theimprovement of explosives analysis. One strategy utilizes laser-inducedbreakdown spectroscopy to identify trace amounts of explosive components[40]. However, this is a destructive technique that does not allowreprieve if not utilized correctly the first time. Much analysis timedoes go into this type of sensor technology to correlate results. Themethod also has to take into account specific factors such as oxygen andnitrogen exposure from the air and also has to make modifications of thetype of gas flow to the instrument in order to obtain spectra fromresidues.

Nanomanipulation-coupled with nanospray ionization provides a straightforward approach to drug and explosives analysis. Crystal extractionprovides an ample signal for detection of ultra-trace amounts of analyteresidue. Sensitivity and selectivity has been demonstrated with thisinstrumental technique along with other hyphenated techniques to thisinstrumentation. The benefits of approach include the elimination ofsample preparation along with immediate analysis. This technique isminimally destructive and offers the ability to utilize optical imagingtechniques to improve the identification of chemical analytes.

Ultra-trace amounts of drug and explosive residues were lifted with acast of a finger impression made from a casting material. The purpose ofthis casting material was to reproduce the friction ridge detail (1902)that would be visibly found on a human finger (FIG. 19). The finger castwas utilized to attract the chemical residues imitating the same methodused for taking fingerprints. The finger cast was slightly saturatedwith a cooking spray oil to imitate the oils naturally secreted from theeccrine glands on the skin's surface of the finger. The chemicalresidues were lifted off a microscopic slide with the saturated fingercast and impressed onto another slide for nanomanipulation-nanosprayionization. The drug and explosive residues were lifted in this mannerto mimic the transfer of residue to the finger that could betheoretically done in a real life scenario.

The drug utilized in this study was powder cocaine in the amount of<0.0050 g. The drug was uniformly mixed with red fluorescent fingerprintpowder (2002) prior to lifting (FIG. 20). UV fluorescence imaging wasdone on this uniform mixture (2002) to investigate the possibleverification of cocaine among a fluorescent powder matrix (not shownhere). Extraction of the drug residue from this uniform mixture (2104)took place with nanomanipulation-coupled with nanospray ionization-massspectrometry (NSI-MS) (2102) following UV fluorescence imaging (FIG.21). The instrument settings for NSI-MS analysis were 2 kV for thevoltage utilizing 10 μL of a 50:50 methanol/water solution with 1%acetic acid for extraction. The MH⁺ peak for cocaine was seen at m/z304.27 on the NSI-MS spectrum (FIG. 22). Spectra were also taken onvarious types of fingerprint powders to obtain a background spectrum foreach powder to examine any interferences to the drug analyte (data notshown). Background spectra were taken from red and orange fluorescentpowders as well as white and black powders. The analysis was done withESI-MS in order to do dilutions of each powder. The mass range was keptthe same as the powder cocaine in order to examine any interferences tothe MH⁺ peak of the drug analyte. The dilutions were kept at a 1/10concentration using a 50:50 methanol/water solution with 1% acetic acid.The ESI-MS spectra of each powder ran for two minutes in positive modeat 5 μL/min with a voltage of 4 kV. An ESI-MS spectrum was also taken ofthe cooking spray oil using the same procedure as the fingerprintpowders (data not shown). The purpose was to examine any interferencefrom the cooking spray oil as well. The spectra for most of the powdersand cooking spray oil show very little indication of interference.

The explosive residues utilized in this study were nitroglycerine (NG)and dinitrotoluene (DNT) stored in methanol. An amount of 10 μL fromeach explosive solution (1 mg/mL) was evaporated off to retrieve thesolid residue particles. The lifting and extraction of these particleresidues followed the same procedure as the powder cocaine. The settingsfor NSI-MS analysis were a voltage of 2.5 kV utilizing 10 μL ofsolution. NSI-MS analysis for the explosive residues ran for 2 min eachin negative mode for NG. The solution mixture containing 5 μL of NH4NO3,10 μL of NH4Cl, and 100 μL of methanol/water solution with 10 μL ofacetic acid was utilized in the extraction process. The purpose of thissolution mixture was to form the adduct peaks with the explosiveresidues to exhibit the presence of the explosives residue due toinstability of the parent ions. For DNT the solvent used was 500 μLmethanol, 500 μL water containing 1 mg of ammonium acetate. 10 μL ofthis solution was used for NSI-MS analysis. The spray voltage was 2.0kV, the scan time was 1 min in the negative mode. NSI-MS spectra wereobtained on each explosive residue prior to the finger print liftstudies to gather standards for comparison.

Example IV NSI-MS for Drug Residue Extraction Coupled to SurfaceEnhanced Raman Scattering (SERS)

The method of residue extraction through electrostatic lifting providesa distinctive mode of performing ultra-trace analysis. These liftsprovide a medium for analyte extraction via NSI-MS. This method ofextraction can be coupled to Raman spectroscopy for supplementalverification of analytes using surface enhanced Raman scattering (SERS).The gold surface used for SERS provides an enhanced effect on peaksignal intensity allowing ultra-trace amounts to be detected moreeffectively. The gold plating of electrostatic lifts allows for atwo-fold method of analysis incorporating improved detection ofultra-trace amounts along with selective probing of the desired analyte.

Raman spectroscopic methods are conventionally utilized for spectralanalysis of small and large compounds in limited amounts. The method isnondestructive in nature and normally requires no sample preparation,making it very efficient. The methods employed provide increasedsensitivity of analyte detection. Many of these Raman spectroscopicmethods have been advanced to provide ultra-trace detection of analytesby modifying the surface which analytes are absorbed or adhered to foranalysis [41]. They have also been coupled to other techniques toimprove overall analysis and sensitivity of detection. Surface enhancedRaman scattering (SERS) has come about to provide improved peak signalintensity from samples analyzed [42]. Enhancement of the Raman signalwith metal substrates has been studied to understand the effect of lightand metal surface interaction as to improve signal intensity of theanalytes [43].

The technique has been implemented in a wide range of analyticalapplications utilizing and experimenting with various metalsubstrates[44]. For single particle analysis, colloidal silver solutionshave been utilized and shown to enhance detection of analytes [45]. SERShas been employed in many types of small and large molecule analysis aswell as various types of applications involving bioselectivespecies[41]. The modification of antigens with gold nanoparticles hasbeen demonstrated with SERS to track antibody production, detection wasexhibited in the femtomolar range [46]. Selective capture of particulardisease causing bacteria by antibody production has been shown usingSERS exhibiting an increased cell readout of 630 to 740 cells/mL in theappropriate buffer solution[47]. Chemical change induction of bacteriaby antibiotics has been monitored using a developed SERS-activesubstrate for analysis [48].

Raman spectroscopic analysis has been incorporated in thecharacterization of various illicit drugs in trace and ultra-traceamounts. The spectroscopic method is versatile in accommodating variousdrug analysis procedures. The detection of cocaine in solid particlemixtures has been performed using Raman spectroscopy implementingprincipal component analysis to facilitate quantitation[49]. This methodof analysis provides a technique for obtaining effective quantitation ofillicit drugs in mixtures utilizing Raman spectroscopy. Cocaine residueon human nail surfaces has been examined with Raman spectroscopy coupledwith confocal microscopy to view the particles through a nail varnishmatrix[50]. This method examined a way to improve analysis byeliminating the matrix effect of the nail varnish toward analysis. Theheighted attention of the SERS method has lead to the research anddevelopment of methods that improve illicit drug analysis. For instance,the examination of gold and silver colloidal suspensions used in SERS todetect amphetamine sulfate has been conducted to compare both in regardsto Raman signal enhancement and surface adsorption[51]. The screening ofecstasy tablets has been successfully performed to detect relativeamounts of MDMA present in various tablets utilizing silvercolloids[52]. The development of improved metal substrates has furtheradvanced the SERS method. Identification of trace amounts of morphine,codeine, and hydrocodone have been detected with SERS using silvernanoparticle substrates to enhance signal intensity and eliminatefluorescence of these compounds[53]. The use of silver halide matriceshas been implemented for the detection of amphetamine in confiscateddrug tablets[54]. The coupling of the SERS method with otherinstrumental methods has been developed as well to advance theimprovement of drug analysis especially when dealing with biologicalmatrices. Trace amounts of narcotic drugs in biological fluids have beendetected with SERS coupled with high performance liquid chromatography(HPLC) as a way to achieve more sensitivity by reducing the matrix[41].Many pharmaceutical companies favor the SERS method over otheranalytical methods due to the overall efficiency and effectiveness ofthe procedure. SERS is ideal for pharmaceutical drug analysis due to thefact that no sample preparation is required and that analysis of samplescan be done in either the solution based or solid form of the analytesin a steadfast manner[55]. The fact that polymorphic behavior of drugscan manifest during spectral analysis provides another reason forpharmaceutical companies to employ SERS[56]. Increased sensitivity canstill be achieved while avoiding this issue. The SERS technique has alsobeen implemented in the procedure for cleaning pharmaceuticalmanufacturing equipment in order to verify cleaning by industrialstandards[57]. The coupling of SERS with nanomanipulation has beenimplemented to provide supplementary characterization of drug analytes.This coupled method has been done to characterize drug analytes on 50 nmgold-plated electrostatic lifts to verify their adherence to the liftvia electrostatic lifting[58]. Spectra of these drug analytes have beencollected in our lab using SERS to provide parallel verification withNSI-MS in other studies.

Electrostatic dust lifting is a method that is utilized in the liftingof prints from many types of surfaces. Surfaces range from a table topto the body of a deceased. The electrostatic lifting process involvesthe use of a high voltage instrument to apply an adjustable voltage tothe metallic side of a Mylar film to lift dust particulates off asurface[59]. Normally the use of the electrostatic lifting process isreserved for crime scene investigations to record prints left on asurface[60]. Results with the electrostatic dust lifter are usuallyachieved with ease and great success as compared with other dust printlifting devices[61]. The technique of lifting dust prints has beenmodified to lift drug particulates in the present invention. Thechemical extraction of drug analytes from these electrostatic lifts arebeing performed with nanomanipulation as opposed to the traditionalphotography utilized in the recording of ridge detail. The electrostaticfilms were coated beforehand with gold to enhance peak signal intensityof the drug analytes present in the soil matrix for the performance ofSERS.

The gold-plating of the electrostatic films for spectral enhancementused in SERS allows for the desired analyte to be detected among abackground matrix. The combination of electrostatic lifting with SERSprovides an inventive approach to performing trace and ultra-traceanalyses of drug particulates. The approach is fast and effective,eliminating the need for sample preparation. This coupled technique hasthe potential to expand to other types of trace work that require solidparticle retrieval and analysis. The combined method can also beemployed with other types of trace methods such as nanomanipulation.

For the electrostatic lifts, Mylar® film (DuPont Wilmington, Del.) wasused as the lifting substrate and a Pathfinder lifter (CSI Equipmentltd. Woburn Sands, England) was used to apply voltage to the filmsduring the lifting procedures. For SERS, gold was deposited on the filmsin 25, 60, and 100 nm layers using chemical vapor deposition. Raman SERSspectra were obtained using a T64000 triple-grating Raman instrument(HORIBA Jobin Yvon Inc. Edison, N.J.), which focused on the samplesurface through a 100× microscope objective. Excitation was accomplishedwith a 532 nm diode-pumped solid-state laser (QED Lasers), attenuated toapproximately 34 mW. Backscattered light was captured with theobjective, guided through the spectrometer and detected using a liquidnitrogen cooled CCD camera.

Caffeine was used to validate the procedure and to probe theeffectiveness of the gold-coated films for use in SERS analysis. First,a small quantity of pure caffeine (<0.003 g) was placed on cleancountertop and a 1.0 in2 sheet of Mylar® was placed over it. Voltage wasthen applied to the film with the lifting apparatus for twenty secondsin order to retrieve the caffeine. The sample was then observed underthe Raman microscope, and particles of approximately 1-3 μm in diameterwere chosen for spectral analysis (unless otherwise noted). Thisprocedure was repeated for each of the gold-plated films.

To test the selectivity of the lifting procedure, small quantities ofcaffeine (<0.003 g) and soil (˜0.020 g, primarily sand) were mixed andthen lifted with Mylar® and the gold-plated films. The films wereweighed before and after each lift in order to determine the amount ofmaterial collected. Raman was used to analyze the lifted material. Thisprocedure was then used to lift and analyze samples of rock cocaine,crystal meth, and ecstasy provided by the University of North TexasPolice Department (Denton, Tex.).

An area of the gold-plated film (60 nm) containing both sand and cocaineparticles was selected for the imaging experiment. Spectra werecollected at 5.0 μm increments in a 60 μm by 80 μm grid and baselinecorrected. Of the resulting spectra, the intensity of the cocaine peakat 1004 cm⁻¹ was used to create a contour plot of the area imaged.

Quantitative amounts of gold (25-100 nm films) were deposited on theMylar® in order to strike a balance between the lifting effect of thesubstrate and the SERS enhancement from the gold. All of the gold-coatedfilms preferentially lifted analyte particles and only trace quantitiesof soil. The sizes of the analyte particles that were lifted varied fromroughly 0.5 mm to 1.0 μm in diameter. Overall, this might suggest thatthe electrostatic properties of the organic analyte particles are moreaccommodating to the gold substrate due to a higher polarizability overthe soil matrix. This effect may also be augmented by the conductivenature of the metal film.

Given the tendency of the gold-coated films to selectively lift theorganic analyte (2304) particles as opposed to the soil matrix (2302)from a solid substrate, support or any solid surface (2308), a two-stepapproach (FIGS. 23A and 23B) (2300) would act to further improve thismethod. In this instance, the print can be lifted using an uncoatedfilm, effectively collecting both the analyte (2304) and matrixparticles (2302) comprising the print. This could be directly followedby a second lift performed by covering the initial lift with agold-coated film (2306) and applying voltage (2310) to collect latentdrug residues. This would provide both an effective print lift, and asubstrate on which to collect and analyze the drug residues via SERS.

FIG. 24 shows the spectra obtained from the caffeine lifted on the fourfilms. On the uncoated Mylar®, only peaks characteristic of the filmitself are observed. The spectrum from the 25 nm gold film displays thelarger peak from caffeine (at 551 cm-1). At 60 and 100 nm gold coverage,the peaks arising from the film have been fully suppressed, and thecaffeine signal is readily identifiable. Full spectra, showing thesuppression of the Mylar® Raman signals, can be seen in FIG. 25 for thecaffeine on Mylar® and the caffeine on the 100 nm gold film. The spectrafrom the caffeine-soil mixture showed no notable differences from thecaffeine standard and have thus been omitted. There was a notableincrease in the mass of lifted particles on the uncoated film, howeverthe mass of particles lifted by gold-coated films still could not bedetermined due to the trace quantities recovered.

FIGS. 26 through 28 show the spectra collected from the mixtures of thedrug samples and soil lifted on the four different films. Again, noanalyte peaks are observed on the uncoated films with the exception of asmall peak at 810 cm-1 in the spectrum for ecstasy. In this case, asignificantly larger particle (˜10 μm in diameter) was chosen foranalysis due to excessive scattering of the laser line and baselinenoise in the spectrum. The spectra for the 25 nm films show no signs ofthe spectral peaks for the film, displaying a successful suppression ofthe background signal. While this is true, the signal for crystal methremains low, showing only the most intense peak in the spectrum near1000 cm-1. The 60 and 100 nm gold films both show enhancements in theRaman spectra of crystal meth and cocaine. The spectrum for ecstasy onthe 60 nm gold film shows a marked reduction in signal strength;however, this is likely to be the result of particle selection and notthe effect of the gold substrate. Overall, the 60 and 100 nm gold filmsshowed the best consistent enhancement of the Raman signal.

FIG. 29 shows the signal-to-noise ratio (SNR) for the four samples oneach of the four substrates. For caffeine, cocaine and crystal meth, theSNR on the uncoated Mylar® is zero, due to the lack of spectral peaksrepresenting the analyte. For the caffeine standard, the 25 nm gold filmshows a marked increase in the SNR. It should be noted that the fullspectrum on the 25 nm film displayed both spectral peaks for caffeineand the Mylar® film. Only at 60 nm were the peaks from the film fullysuppressed. For the three illicit drug samples, it can be seen that theSNR increases with presence of thicker gold layers (with the exceptionof the aforementioned MDMA sample).

FIGS. 30A-30C show both the microscope image (3002) of the section usedin the imaging study (FIGS. 30A and 30B) and the resulting Raman map(3006) of the surface (FIG. 30C). Of the particles visible on thesurface, three can easily be identified as rock cocaine (3004) based onthe Raman spectra of the surface. This method of screening lends itselfto quick identification of the particles and can be followed withadditional particle-specific analysis such as nanoextraction andidentification with NSI-MS.

The coupling of the electrostatic lifting method with gold-coated Mylar®films has been shown to be an effective process in the analysis ofultra-trace drug residues. By selectively lifting analyte particles overthe matrix, the overall time for microscopic screening of the liftcontents is effectively reduced. This also lends itself toanalyte-specific extraction while keeping the original lifted printintact. In addition to this, the gold coatings on the films provide aneffective substrate on which to perform SERS analysis. The analytespectra display significant signal increases with the presence of thegold films, and the peaks arising from the film substrate are reduced oreliminated.

Example V Direct Identification of Lipid Composition in Lipid Droplets(LDs) by Nanospray Mass Spectrometry

LDs are organelles that are specialized for the storage of neutrallipids and as such provide energy-rich reserves in all cellularorganisms[62]. Understanding LD ontogeny is of major importance to humanphysiology; on the one hand, seed oils packaged in LDs make up a growingproportion of daily caloric intake in most diets around the world, andon the other hand, the regulation of lipid storage and mobilizationunderlies significant human health issues: obesity, diabetes andcardiovascular disease.

Although storage is considered the principal role of neutral lipids, LDsin nonfat storing tissues recently have become more appreciated fortheir dynamic nature and functional roles independent of storage. Theseroles include acyl reserves for phospholipid recycling [63], lipidsignaling [64], membrane trafficking [63, 65], inflammation and cancer[66], and host-pathogen interactions [67, 68]. These various functionsattributed to LDs vary with cell type and likely are manifested bydifferences in droplet composition. The basic structural model of LDs inplant seeds provides a thermodynamically stable organization that isthought to be conserved throughout eukaryotes, although the nature ofthe lipids and proteins associated with droplets varies with cell/tissuetype. The structure describes a neutral lipid core (triacylglycerols inplant seeds and/or steryl esters in other organisms or cell types)surrounded by a phospholipid monolayer with specific proteins associatedwith the LD surface[69]. Although the endoplasmic reticulum isconsidered by most to be the major cellular location for LD biogenesis,droplets associate frequently with other subcellular compartments,presumably to carry out unique functions [70].

The recent emphasis on studying the formation and turnover of theseorganelles and the importance of this compartment to general cellularphysiology has prompted the development of advanced analytical tools forthese organelles. Visualization of cytosolic LDs has commonly beencarried out by conventional light microscopy and confirmed byhistochemical and/or fluorescent neutral lipid specific stains (e.g.Sudan III, Nile Red, and BODIPY derivatives[71-73]. Electron microscopy,such as transmission electron microscopy or freeze-fracture and lowtemperature scanning electron microscopy, have supported the descriptionof the fine ultrastructure of LDs within various plant and mammaliantissues yielding information on structural variability among anassortment of mutants and under a range of environmentalconditions[74-77]. More recently, third-harmonic generation microscopy[78] and high resolution, nonresonant confocal Raman microscopy [79]have been developed to selectively image unstained LDs unveiling novelinteractions in complex cellular environments. In combination withtwo-photon and second-harmonic generation microscopy, third-harmonicgeneration microscopy offers three-dimensional spatial resolution thatcan be used to visualize LDs for long periods. Raman-based microcopy caneven provide some molecular composition information for LDs withinsingle cells.

The rapidly developing field of lipidomics has led to a renewed effortto analyze triacylglycerol (TAG) prevalence and composition within LDsby mass spectrometric approaches [63]. Many studies have now detailedthe complex fragmentation patterns for the complete structuralelucidation and quantification of TAGs [80-82]. Supported by advances inbioinformatics (LIPID MAPS) [83], improvements in mass spectrometry, andavailability of unique purified standards, it is now feasible to achievecomprehensive lipid identification and quantification directly fromcomplex mixtures. For lipidomics applications, lipids most often areextracted from tissues or cell lines in organic solvents, losing thespatial information of lipid organization within the original sample.Others have combined mass spectrometry with microscopy approaches suchas MALDI-MS [84], secondary ion MS (SIMS) [85], and desorptionelectrospray ionization (DESI)-MS [86] to preserve spatial context withcomposition information, but the resolution currently remains at thecellular/tissue level, and the compositional analysis is limited andincomplete.

Here, we have developed a novel technique for direct organelle massspectrometry (DOMS) that couples direct visualization with detailed massspectrometric analysis of organelles. A multifaceted nanomanipulator,previously demonstrated to extract peptides from a single bead [87] andextract and analyze trace fiber analytes [88], was equipped with glassnanospray emitters prefilled with organic solvent capable of extractingthe lipid contents out of LDs. This approach is illustrated herein withLDs from diverse plant sources (Gossypium hirsutum and Arabidopsisthaliana). The inventors demonstrate the capability to directly samplepopulations of purified LDs as well as perform single-organelle massspectrometry and lipid characterization. To illustrate the utility ofthis approach, the inventors show a compositional shift in TAG profilesin LDs purified from modified oleic cottonseed lines previouslygenerated by the inventors (dominant-negative expression of Bnfad2 [89,90], the presence of cyclic fatty acids in TAGs of cotton root LDs, andthe molecular comparison between Arabidopsis seed and leaf LDs. Thesenew approaches complement existing analytical and cell biologytechniques and can be extended to the analysis of LDs and organellesfrom other organisms. This approach will help facilitate new studiesabout LD heterogeneity and the molecular nature of subcellularcompartments in cellular systems.

Plant Growth Conditions: Cottonseeds were propagated underair-conditioned greenhouse conditions at 30° C. and supplemented withsodium vapor lamps to extend day length to 16 h. Opened bolls wereharvested, and seeds were delinted in a table top, 10-saw laboratorygin. Seeds were from cultivar Coker 312 (nontransgenic) or were fromtransgenic lines (T5 generation) in the Coker 312 background, expressinga nonfunctional allele of the Brassica napus Δ12-desaturase (Bnfad2)under the control of a seed-specific promoter [91, 92]. These seedsdisplayed reduced oil, elevated oleic acid phenotype relative to Coker312 seeds [91, 92]. Intact embryos (mostly cotyledon tissues) wereharvested from desiccated seeds following seed coat removal. Roots werecollected on ice from germinated cottonseeds (cv. Coker 312) grown inthe dark at 30° C. for 48-72 h. Arabidopsis wild type (Col 0) seeds wereobtained from the Arabidopsis stock center at Ohio State University andpropagated in house. Plants were grown in soil at 21° C. in a 16-hlight/8-h dark cycle (between 45 and 65 mol/m2/s).

Imaging Lipid Droplets in Situ: LDs were imaged by confocal scanningfluorescence microscopy using BODIPY 493/503 to selectively visualizeLDs in situ. All tissues were fixed in 4% w/v paraformaldehyde in 50 mMPIPES, pH 7.0, and stained with 1-10 μg/mL BODIPY 493/503. For BODIPYimaging, excitation of BODIPY was at 493 nm. Emission wavelength forBODIPY-stained LDs was 520 nm, exposed for 0.4-10 s with no gain. Inleaves, chloroplast autofluorescence was acquired with an excitation of493 nm and an emission wavelength at 692 nm exposed for 0.4 s. Imageswere acquired with a Zeiss 200 M optical microscope fitted with a CSU-10Yokogawa confocal scanner (McBain Instruments) and captured with adigital camera (Hamamatsu, Phoenix, Ariz.). LD morphology andlocalization were characterized using the McMaster Biophotonics Facilityand NIH Image) software (version 1.43T).

Lipid Droplet Purification: LDs from unfixed embryos of maturecottonseeds were isolated and purified in 100 mM Tris-HCl, 600 mMsucrose, 10 mM KCl, 1 mM EDTA, essentially as described by Chapman andTrelease [91] and based on methods developed by Huang [92]. Embryos werechopped into ˜1-mm pieces with a razor blade in ice-cold buffer andpurified through a sequential series of three floatations through 500 mMsucrose at 10,000×g (Sorvall SS-34 rotor or HB-6 rotor in a Sorvall RC5C centrifuge, or in a Centronix Microcentrifuge 1236V for smallersample sizes). LDs were purified in a similar manner from cotton roots(at least 500 mg fresh weight) of 48-72-h-old seedlings, Arabidopsisleaves of 40-day-old plants (˜8 g of fresh weight), or Arabidopsis seeds(10 mg). LDs from Arabidopsis leaves are few in number, and a finalultracentrifugation step (TLA-100 rotor at 100,000×g for 1 h, with aBeckman TL100 ultracentrifuge) helped to enrich these organelles in thetop layer. For some studies, 50 mM PIPES-NaOH, pH 7.0, buffer wasemployed for homogenization and floatation. For all purifications, thefat pads were carefully collected with a spatula, and the LDs weresuspended in buffer on ice. LDs were stained with 0.1-1 μg/mL of BODIPY493/503 in 50 mM PIPES buffer.

Nanomanipulator Work Station: The Biometric L200 nanomanipulator workstation developed by Zyvex (Richardson, Tex.) and the present inventorscombined four nanopositioners (3102) with a piezo voltage source and aPM 2000B programmable four-channel pressure injector (3104) (MicrodataInstruments, South Plainfield, N.J.) situated on an inverted microscopestage (3110) (TE2000U, Nikon, Melville, N.Y., diagrammed in FIG. 31A andFIG. 31B). The nanomanipulator has two modes of motion along the x, y,and z axes. The fine mode has 100 microns of travel in the x and z axisand 10 microns of travel in the y axis with 3.4 nm resolution controlledby piezoelectric crystals. The coarse mode has 12 mm of travel in the xaxis and z axis and 28 mm of travel in the y axis with 100-nmresolution. The positioners consist of end effectors made up of sixisolated, low impedance electrical connections and two glass capillaryattachments. The end effectors are used for manipulation and, if needed,low impedance electrical characterization. Probes and capillaries (3106)attached to the positioners can be manually landed onto the sample andmanipulated electronically using a joystick (3112) to control theposition of the end effector. The capillaries (3106) are connected byTeflon tubing to a PM 2000B programmable four-channel pressure injector.LDs were visualized by epifluorescence, bright field, or differentialscanning interference optics during collection (Nikon NIS elements), andthe lipids (3108) were microextracted in 10 mM ammonium acetate inchloroform:methanol (1:1, v/v) in the capillary before MS. Forsingle-lipid droplet analysis, the extraction solution was spiked with aTri 15:0 TAG standard at a final concentration of 2 μM.

Nanospray Mass Spectrometry: A Proxeon nanospray source (ProxeonBiosystems, Odense, Denmark) was mounted on a Thermo LCQ Deca XP Plusquadrupole ion trap (Thermo Fisher Scientific). The lipids in thenanospray emitters (New Objective (Woburn, Mass.) Econo12 PicoTip™Emitter platinum-coated or PROT12 break-to-open platinum emitters (tipopening diameters were 1±0.2 μm and 1-10 μm, respectively) weresubjected to typical ion source conditions consisting of a potential of0.8 to 1.5 kV, capillary temperature of 200° C., and capillary voltageof 3.0 V. Mass spectra were acquired using the LCQ Tune software programin the positive ion mode with three microscans and a continuousacquisition time. The mass spectra were analyzed with the XCalibursoftware package (version 2.0). Tandem mass spectrometry (MS/MS)analyses were used to confirm the identity of the triacylglycerols by acharacteristic diacylglycerol fragment. Tandem spectra were acquired inpositive ion mode with a typical isolation width of 3.0 m/z, normalizedcollision energy of 35%, activation Q of 0.250, and an activation timeof 30 ms. Quantitative estimates of molecular compositions werecalculated in Microsoft Excel with in house algorithms integrating peakareas, correcting for isotopic overlap, and when available normalizingto internal standards.

Conventional Lipid Extraction: Total lipids were extracted from LDspurified from mature embryos of desiccated seeds (cv. Coker 312, withoutand with a Brassica FAD2 nonfunctional allele) using the method of Blighand Dyer [93]. Total lipid extracts were dissolved in 10 mM ammoniumacetate in chloroform:methanol (1:1, v/v) and characterized by nanospraymass spectrometry or converted to fatty acid methyl esters and analyzedby gas chromatography equipped with a flame ionization detector. [91]

Fatty Acid Methyl Ester Preparation and Analysis: Additional total lipidextracts of the purified LD fractions were converted into fatty acidmethyl esters. Fatty acid methyl esters were prepared bytransesterification with 1 N methanolic HCl at 85° C. for 2 h. Aheptadecanoic acid (C17:0) standard was added to aid quantification.Fatty acid methyl esters were separated on a 30 m×0.25 mm inner diameterSupelcowax 10 fused silica capillary column on a HP 5890 Series II Plusgas chromatograph with an initial oven temperature of 200° C. increasingat a rate of 1.3° C./min to a final temperature of 230° C.

Electrospray Mass Spectrometry: Because the ion trap is limited in itsability to carry out precursor-product scans, additional acyl chaininformation was derived from analysis of total lipid extracts of LDfractions purified from cotton (seed, root) and Arabidopsis tissues(seed, leaf) using a Waters Micromass Quattro Ultima triple quadrupolemass spectrometer (Waters, Milford, Mass.). Typical scanning conditionsfor a direct infusion rate of 10-20 μl/min were carried out in positiveion mode with a 3-3.5 kV spray voltage, 40 V cone voltage, and a scanrange of 750 to 1000 m/z. The desolvation and source temperatures weremaintained at 200 and 80° C., respectively, and the desolvation and conegas flows were set at 300 and 80 liters/hr, respectively. Tandem scans(MS/MS), whether detecting the precursor ions that lost a particularacyl chain in neutral loss mode or single precursor-product species,were performed with collision energy of 30 V with a scan range from 400to 1000 m/z.

Direct Organelle Mass Spectrometry: Interfacing direct organellesampling with nanospray mass spectrometry required the incorporation ofnanospray emitters that could control the liquid flux at the tip openingin contact with the sample of interest while also sufficientlyconcentrating the extracted lipids to detect their composition with highresolution. The development of a nanomanipulator apparatus (FIG. 31)[87] coordinated the positioning of up to four emitters in athree-dimensional plane situated on the stage of an inverted lightmicroscope for maximum working distance. Emitters, prefilled with anorganic microextraction solution that served the dual purpose ofextracting the lipids from the droplets and facilitating the formationof nanospray droplets, were positioned adjacent to purified LDs (FIG.31A). Direct interfacing of the emitter with a dynamic pressure injectorprovided the sensitivity necessary to fill nanoliter volumes (usuallybetween 5-30 psi of fill pressure) and selectively capture individualorganelles. After sampling LDs, the emitters were remounted onto thenanospray apparatus (FIG. 31B) for chemical analysis.

Lipid Droplet Characterization: To test the DOMS approach, LDs werepurified from mature cotton embryos (cv. Coker 312) due to theirabundance within seed tissues, relative ease of purification and theknown composition of TAGs in standard cottonseed oil [33]. These LDs,suspended in ˜100 μL, were somewhat variable in size (˜0.5-2.0 μmdiameter) (FIG. 31A) but retained similar spherical morphology to thatobserved in situ. A random sampling of approximately two dozen LDsproduced a high resolution spectrum of TAGs (FIG. 32A). The TAGmolecular species under these conditions were of sufficientconcentration to analyze by MS/MS and confirm/assign acyl composition(FIGS. 32B and 32C). Tandem MS of TAG precursor ions producediacylglycerol product ions that lead to a high confidence of acyl chainidentification [94]. The TAG acyl chain distribution in purifiedcottonseed LDs primarily consisted of linoleic (18:2), oleic (18:1), andpalmitic acids (16:0).

Most LDs share a similar spherical morphology despite some significantalterations in the composition of their neutral lipid core orsurrounding phospholipid/protein-lined monolayer [69, 73, 90]. However,the size of LDs is much more variable depending on tissue type [62]and/or metabolic state [73, 90]. Expression of a Brassica nonfunctionalallele of a δ-12 fatty acid desaturase (Bnfad2) in a Coker 312 wild typebackground produces fewer and larger LDs in cotyledons (FIGS. 33A and33B) [90]. Purification of these LDs was verified by bright field (FIGS.33C and 33D) and epifluorescence microscopy (stained with a neutrallipid specific fluorescent dye, BODIPY 493/503) (FIGS. 33E and 33F).Direct sampling of these Bnfad2 LDs showed a distinct shift towardincreased oleic acid (18:1) acyl chain distribution within TAG at theexpense of linoleic acid (18:2) (FIG. 33G) that was confirmedqualitatively and quantitatively with conventional chemical extractionsof total seed lipids (FIGS. 34A and 34B) and analysis of total fattyacid content by gas chromatography equipped with a flame ionizationdetector, consistent with suppression of endogenous oleic aciddesaturation by FAD2 (dominant-negative mutation). Hence, with thisapproach, it was possible to demonstrate directly a change in molecularcomposition at the organelle level in these metabolic mutants.

Sampling multiple LDs resulted in sufficient TAG concentrations withinthe nanospray emitters to allow for detailed compositional informationto be acquired through tandem MS. The overall variability in TAGcomposition from sampling small, random populations of cottonseed LDs(10-25 droplets per sample) was relatively low (FIGS. 34C and 34D).Direct visualization and sampling of a single Bnfad2 LD (FIG. 34B)required fine tuning the filling conditions to prevent multiple LDs fromentering the tip and obscuring the final suspension of LD.

To assess the heterogeneity of individual LDs isolated from cottonseedembryos, LDs were sampled individually for both the Coker 312 and Bnfad2(Tables 2 and 3 and FIGS. 35A-35C). Representative TAG spectra fromsingle Coker 312 (FIG. 35B, top) and single Bnfad2 (FIG. 35B, bottom) LDshowed identifiable TAG profiles of single LD. There was a substantialdifference in mol % of TAG species (normalized to Tri 15:0 internalstandard) among individual LD, both within the Coker 312 seeds and theBnfad2 seeds (Tables 2 and 3). The average TAG molecular composition(FIG. 35C; Tables 2 and 3) of seven individual LDs combined weredistinctly different from one another (Coker 312 versus Bnfad2), and theaverage composition of each variety approached that of the compositionvarieties when multiple LDs were sampled together. However, there wasconsiderable heterogeneity among purified seed lipid droplets,suggesting a complexity in the biogenesis of LD not able to beappreciated until now. In other words, the overall average lipidcomposition of these seeds comes from discrete LD packages with variableTAG profiles. This is new information about LDs that would not bepossible were an individual droplet not able to be sampled directly byMS. These results indicate that this technique of DOMS can be used toassess organelle heterogeneity by sampling single organelles.

One advantage of this DOMS approach would be to distinguish by molecularcomposition between LDs of similar morphology. Cyclic fatty acidsaccumulate in root tissues of cotton seedlings during the early growthstages and include malvalic acid (8,9-methylene-8Z-heptadecenoic acid,32.6% of total fatty acid), sterculic acid(9,10-methylene-9-octadecenoic acid, 11.0%), and dihydrosterculic acid(9S,10R-methyleneoctadecanoic acid, 0.6%) (83, 35). These cyclic fattyacids are enriched in the TAG fractions and presumed to be packaged intoLDs in root cells. LDs in root cells (visualized by BODIPY-specificfluorescent staining) appeared to be either localized in clusters aroundthe nucleus or dispersed throughout the cytosol in different root cells(FIG. 36A). Isolation and purification of these LDs did not affect theirsize and morphology (FIG. 36B), and this morphology was similar to theLDs purified from cotyledons of seed tissues (FIG. 33C). TAG profiles inroot LDs were distinct from those derived from cotyledon tissues ofseeds (FIG. 36C), and indeed, these root TAG were enriched in cyclicfatty acids with sterculic and dihydrosterulic acids on one or two acylchains (confirmed by tandem MS, FIGS. 37 B and C). Although malvalicacid is known to be prevalent in roots by gas chromatographic studies[95], here by MS, it was not distinguishable from linoleic acid (FIG.37A) in the purified droplets (both fatty acids have a molecular mass of280.45 g/mol). Nonetheless, it was possible to directly distinguish LDsof differing molecular composition that were otherwise indistinguishableby morphology, illustrating that DOMS might be used to assess organelleheterogeneity within a given tissue or cell type.

TABLE 2 Heterogeneity of TAG molecular species in Coker 312-purifiedseed lipid droplets P, 16:0-palmitic acid; O, 18:1 oleic acid; L,18:2-linoleic acid. TAG molecular composition PPL^(a) PPO PLL PLO POOLLL LLO LOO OOO mol % LD1 15.9 4.5 19.8 14.8 9.8 11.0 10.9 6.1 7.1 LD29.1 5.7 18.5 11.5 10.4 15.2 13.0 10.4 6.3 LD3 14.7 9.1 16.0 12.4 9.0 9.810.9 8.4 9.6 LD4 17.2 4.3 27.9 14.2 4.9 13.1 9.7 5.4 3.2 LD5 16.7 3.826.0 14.1 6.3 12.3 10.1 6.0 4.7 LD6 13.0 2.6 26.9 13.4 6.5 16.3 10.5 6.14.6 LD7 7.2 4.2 16.6 13.2 9.1 16.6 13.7 11.4 8.0 Mean 13.4 4.9 21.7 13.48.0 13.5 11.3 7.7 6.2 S.D. 3.9 2.1 5.1 1.2 2.1 2.6 1.5 2.4 2.2 ^(a)TAGacyl chains are sn-nonspecific.

TABLE 3 Heterogeneity of TAG molecular species in Bnfad2-purified seedlipid droplets P, 16:0-palmitic acid; O, 18:1 oleic acid; L,18:2-linoleic acid. TAG molecular composition PPL^(a) PPO PLL PLO POOLLL LLO LOO OOO mol % LD1 17.9 14.0 7.0 16.2 22.7 1.6 4.0 8.8 7.7 LD213.0 8.3 13.2 22.3 15.4 6.5 10.3 7.9 3.1 LD3 13.2 9.4 11.4 21.4 20.4 5.08.5 6.9 3.8 LD4 14.9 11.6 9.2 19.8 22.8 3.7 5.5 6.5 6.0 LD5 13.6 7.514.1 20.4 18.5 5.1 10.7 8.3 1.7 LD6 11.8 10.5 10.8 22.8 20.3 4.1 7.8 9.03.0 LD7 16.0 15.0 10.6 17.1 20.7 4.5 7.8 7.7 0.7 Mean 14.3 10.9 10.920.0 20.1 4.4 7.8 7.9 3.7 S.D. 2.1 2.8 2.4 2.5 2.5 1.5 2.4 0.9 2.4^(a)TAG acyl chains are sn-nonspecific.

A single A. thaliana seed (˜15-25 μg) provides more than enough LDs fordirect organelle mass spectrometry (FIG. 38A), and these seed LDs can bepurified by a rapid two-step procedure (FIG. 38F) reducing the time fromLD purification to determination of LD TAG composition to less than anhour. On the other hand, the rosette leaves (˜1 mg of dry weight) of40-day-old Arabidopsis plants contain very few LDs per cell (FIGS. 38 Cand E), and much more tissue was required for purification of LDs fromleaves. DOMS showed that the seed LDs contained characteristic20:1/eicosenoic fatty acids in their TAGs, whereas the LDs from leavesinstead contained more 16:3 and 18:3 fatty acids that are mostcharacteristic of leaf acyl lipids (FIG. 38B) [96]. LDs in leaves (FIG.38C), although similar in morphology to those in seeds (FIG. 38D),likely have a function different from the long term storage function ofTAGs in seeds for germination and seedling establishment. Profilingorganelles by DOMS from different tissues and metabolic contexts willprovide new insights into how cellular and subcellular heterogeneitycontributes to cellular function, which have been questions difficult toaddress directly until now.

The development of the L200 nanomanipulator supports a variety ofpotential analytical techniques at the cellular and subcellular level.Here, we illustrate some of these capabilities by the directvisualization of LDs derived from various cell types coupled withdetailed chemical analysis. Conventional lipid profiling by MS, althoughdetailed and capable of resolving highly complex compositions ofmolecules at a range of endogenous concentrations, relies on totalextractions of lipid molecules from organs, tissues, or cell types,resulting in a rich mixture of compounds that lose all spatial context.Here, the DOMS facilitates a complete, comprehensive lipidomicsprofiling while maintaining organellar identity of the sample source.Although seed LDs contain mostly TAG molecules, there are also a smallproportion of phospholipids and proteins (e.g. ˜97% TAG, 1%phospholipid, 2% protein in a 1-μm diameter LD) [69]. Although only TAGmolecules were detected with DOMS, it is likely that ion suppressioneffects prevented the detection of phospholipid with the ion trap MS. Itis possible through instrument modifications that interfacing DOMS witha triple quadrupole MS might facilitate the detection of phospholipidand/or proteins.

The technique presented hereinabove is versatile and could be combinedfor the lipid profiling of other subcellular components and combinedwith microscopic analysis serve as a means of evaluating detailedmolecular changes at the organellar level to address arrange ofbiological questions intractable until now. For example, considering thelow variability of sampling multiple LDs, it was surprising to uncoversignificant TAG compositional heterogeneity when sampling individualLDs, pointing to complexity at the subcellular level in packaging TAGinto lipid droplets, that until now had not been considered in models ofLD biogenesis. Furthermore, it is possible to envision this approachexpanded to support broad-based MS analysis of other macromolecules inorganellar samples, such as proteomic or general metabolomic studies,placing this DOMS approach at the forefront of biochemical analysis withmicroscale resolution. Although the positioning resolution of the L200surpasses many commercially available nanomanipulators, the relativeease of combination of a standard robotic-controlled microscope stagewith conventional light microscopy and standard MS instruments(nanospray source mounted on an ion-trap or triple quad MS) provides ahigh level of flexibility to achieve diverse and specialized systems[87, 88]. In this case, only a single end effector holding a glassnanospray emitter was necessary for sampling purified LDs; however,there are multiple ports on the L200 that could be used as molecular“tweezers” and low impedance electrical positioners [87] that might beadvantageous for handling and/or sampling cellular constituents onstage, either from purified populations as was shown here, or perhapseven in situ by microdissection of whole tissue samples. Currently thenanospray emitters are not designed to penetrate the thick cell walls ofplant tissues and permit selection of organelles in situ. In the future,it might be possible to use some combination of laser microdissection orcell wall digestion to gain access to organelles within tissue samples,and advances in sample preparation will expand the uses of DOMS.

Recent advances in imaging MS (MALDI-MS [84], DESI-MS [86], SIMS [79,85], and Raman spectroscopy [79] have acquired compositional informationin association with spatial distribution in biological specimens.Unfortunately, the limited resolution of MALDI-MS (typical lateralresolution of 25-100 μm [84]) and DESI-MS (typically only 250 μm [86])makes it impossible to resolve the compositional information of singleorganelles. Although Raman spectroscopy approaches afford some chemicalcompositional information at high spatial resolution in situ, thisinformation is limited to gross chemical information, such as confirmingneutral lipid classes or overall saturation of acyl chains. Asignificant advantage of imaging MS is acquiring chemical information insitu. However, significant tissue preparation is often required that canadversely affect quantitative accuracy and compositional distribution[84]. For example, the quantitative accuracy of MALDI-MS and TOF-SIMS issignificantly affected by sample preparation necessary to withstandvacuum pressure and ion generation energies [86]. The DOMS method of thepresent invention can derive information at a single organelle (LD)level with more comprehensive chemical compositional information than ispossible by any current MS imaging approach that relies on directionization properties of molecules from a surface. Indeed the DOMSapproach might be modified to be combined or to verify various in situimaging techniques and together help to generate more complete chemicalmaps of cells and subcellular compartments than is currently available.The DOMS approach developed and described herein has the potential to beapplied to diverse areas of cell biology and address many questions. Inspecific applications toward LDs, progress has been made inunderstanding the mechanisms of lipid production, packaging intocytosolic LDs, and physiological roles of LDs.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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What is claimed is:
 1. A method for identifying, detecting, analyzing orcombinations thereof of one or more analytes from a substrate comprisingthe steps of: providing the substrate comprising the one or moreanalytes to be detected, wherein the substrate is fixedly mounteddirectly or indirectly on an inverted microscopic stage; providing anextraction system for an extraction of the one or more analytes from thesubstrate followed by a transfer to a detection system, wherein thedetection system is coupled to the extraction system, wherein theextraction system comprises: a workstation comprising a plurality ofmoveable nanopositioners, wherein the nanopositioners are capable ofmovement in a X direction and in a Y direction and in a Z direction withrespect to the fixedly mounted substrate, wherein the nanopositionershold one or more probes, grippers, capillary tips or any other suitableaccessory for an extraction and transfer of a liquid phase and thenanopositioners permit detection of analytes at the cellular orsubcellular level; a pressure injector for a delivery of a pressurizedextraction solvent through a capillary tip, wherein the capillary tip isplaced in close proximity to the substrate wherein the pressurizedextraction solvent contains at least one extraction solvent that isspecific for an analyte on the substrate that is to be dissolved; avoltage source; a joystick or a digital controller for controlling themovement of the nanopositioners; and a mass spectrometer that is offline or is connected to the extraction system for receiving the one ormore dissolved analytes transferred by the extraction system; dissolvingthe one or more analytes by an injection of the extraction solvent ontothe substrate; aspirating the dissolved analytes by the one or morecapillary tips; injecting the aspirated analytes into the massspectrometer to obtain a mass spectrum; and identifying the one or moreanalytes by a m/z ratio in the mass spectrum.
 2. The method of claim 1,further comprising the step of determining a concentration of the one ormore analytes on the substrate.
 3. The method of claim 1, wherein theone or more analytes are selected from the group consisting ofindustrial, chemical, and biological materials, biological cells,paints, inks, pigments, dyes, illicit drugs, pharmaceuticals, proteins,and combinations and modifications thereof.
 4. The method of claim 1,wherein the substrate is selected from the group consisting of fibers,clothing, hair, paper, money, computer chips, treated wood, laminate,metal, manipulated surfaces including mylar electrostatic lift films,plastic, and combinations and modifications thereof.
 5. The method ofclaim 1, wherein the mass spectrometer is equipped with a nanospraysource.
 6. The method of claim 1, wherein the substrate is mounted on anon-inverted microscopic stage.
 7. The method of claim 1, wherein themethod is used for analysis of documents, analysis of artworks, illicitdrug detection, forensic and toxicology studies, and combinations andmodifications thereof.
 8. The method of claim 1, wherein the extractionsolvent comprises water, polar organic and inorganic solvents, mixturesof polar and non-polar solvents, and combinations and modificationsthereof.
 9. A system for identifying, detecting, analyzing orcombinations thereof of one or more analytes from a fixed substratecomprising: an inverted or non-inverted microscopic stage capable ofholding the substrate comprising the one or more analytes; a workstationcomprising a plurality of moveable nanopositioners, wherein thenanopositioners are capable of movement in a X direction and in a Ydirection and in a Z direction with respect to the fixed substrate,wherein the nanopositioners hold one or more probes, grippers, capillarytips or any other suitable accessory for an extraction and transfer of aliquid phase and the nanopositioners permit detection of analytes at thecellular or subcellular level; a pressure injector for delivering apressurized extraction solvent through a capillary tip, wherein thecapillary tip is placed in close proximity to the substrate wherein thepressurized extraction solvent contains at least one extraction solventthat is specific for an analyte on the substrate that is to bedissolved; a voltage source; a joystick or a digital controller forcontrolling the movement of the nanopositioners; and a mass spectrometerthat is off line or is connected to the extraction system for receivingthe one or more dissolved analytes transferred by the extraction systemand for the identifying the one or more analytes by a m/z ratio from agenerated mass spectrum.
 10. The system of claim 9, further comprisingthe step of determining a concentration of the one or more analytes onthe substrate.
 11. The system of claim 9, wherein the one or moreanalytes are selected from the group consisting of industrial, chemical,and biological materials, cells, organelles, paints, inks, pigments,dyes, illicit drugs, pharmaceuticals, proteins, and combinations andmodifications thereof.
 12. The system of claim 11, wherein the illicitdrug is cocaine.
 13. The system of claim 11, wherein the organelle is amitochondria.
 14. The system of claim 9, wherein the substrate isselected from the group consisting of fibers, clothing, hair, paper,money, computer chips, treated wood, laminate, metal, manipulatedsurfaces including mylar electrostatic lift films, plastic, andcombinations and modifications thereof.
 15. The system of claim 9,wherein the mass spectrometer is equipped with a nanospray source. 16.The system of claim 9, wherein the system is used for analysis ofdocuments, analysis of artworks, illicit drug detection, forensic andtoxicology studies, and combinations and modifications thereof.
 17. Thesystem of claim 9, wherein the extraction solvent comprises water, polarorganic and inorganic solvents, mixtures of polar and non-polarsolvents, and combinations and modifications thereof.
 18. A method forcompositional analysis, concentrating one or more analytes orcombinations thereof comprising the steps of: providing the one or moreanalytes in a first liquid phase on a substrate fixedly mounted directlyor indirectly on an inverted microscopic stage; providing an extractionsystem for an extraction of the one or more analytes from the substratefollowed by a transfer to a detection system, wherein the detectionsystem is coupled to the extraction system, wherein the extractionsystem comprises: a workstation comprising a plurality of moveablenanopositioners, wherein the nanopositioners are capable of movement ina X direction and in a Y direction and in a Z direction with respect tothe fixedly mounted substrate, wherein the nanopositioners hold one ormore probes, grippers, capillary tips or any other suitable accessoryfor an extraction and transfer of a liquid phase and the nanopositionerspermit detection of analytes at the cellular or subcellular level; apressure injector for delivering a pressurized extraction solventthrough a capillary tip, wherein the capillary tip is placed in closeproximity to the substrate wherein the pressurized extraction solventcontains at least one extraction solvent that is specific for an analyteon the substrate that is to be dissolved, wherein the extraction solventis immiscible or partially miscible with the first liquid phase; avoltage source; a joystick or a digital controller for controlling themovement of the nanopositioners; and a mass spectrometer that is offline or is connected to the extraction system for receiving the one ormore analytes transferred by the extraction system; solubilizing orpartitioning the one or more analytes between the first liquid phase andthe extraction solvent by an injection of the extraction solvent ontothe substrate; aspirating a mixture of the solubilized or partitionedanalytes by the one or more capillary tips; mixing the aspirated mixtureby a movement of the mixture in the capillary tube; injecting theaspirated analytes into the mass spectrometer to obtain a mass spectrum;and identifying the one or more analytes by a m/z ratio in the massspectrum.
 19. The method of claim 18, further comprising the step ofdetermining a concentration of the one or more analytes in thesubstrate.
 20. The method of claim 19, wherein the step of determiningthe concentration is done by an injection of an internal standard,wherein the internal standard is a pure sample or a derivative of theanalyte having a similar mass.
 21. The method of claim 18, wherein theone or more analytes are selected from the group consisting ofbiomolecules, lipophilic compounds, oils, triacylglycerols, vegetableoil products, cottonseed oil, serum, proteins, antibodies, andcombinations and modifications thereof.
 22. The method of claim 21,wherein the oil is cottonseed oil.
 23. The method of claim 21, whereinthe serum is a rabbit serum.
 24. The method of claim 18, wherein themass spectrometer is equipped with a nanospray source.
 25. The method ofclaim 18, wherein the extraction solvent comprises water, polar organicand non-polar organic solvents, mixtures of polar and non-polarsolvents, and combinations and modifications thereof.
 26. The method ofclaim 25, wherein the extraction solvent is a 1:1 mixture of chloroformand methanol, comprising about 2% acetic acid.
 27. A system forcompositional analysis or concentrating one or more analytes orcombinations thereof from a fixed substrate comprising: an invertedmicroscopic stage mounted with or capable of holding the one or moreanalytes in a first liquid phase; an extraction system for an extractionof the one or more analytes from the substrate followed by a transfer toa detection system, wherein the detection system is coupled to theextraction system, wherein the extraction system comprises: aworkstation comprising a plurality of moveable nanopositioners, whereinthe nanopositioners are capable of movement in a X direction and in a Ydirection and in a Z direction with respect to the fixed substrate,wherein the nanopositioners hold one or more probes, grippers, capillarytips or any other suitable accessory for an extraction and transfer of aliquid phase and the nanopositioners permit detection of analytes at thecellular or subcellular level; a pressure injector for delivering apressurized extraction solvent through a capillary tip, wherein thecapillary tip is placed in close proximity to the substrate wherein thepressurized extraction solvent contains at least one extraction solventthat is specific for an analyte on the substrate that is to bedissolved, wherein the extraction solvent is immiscible or partiallymiscible with the first liquid phase; a voltage source; a joystick or adigital controller for controlling the movement of the nanopositioners;and a mass spectrometer that is off line or is connected to theextraction system for receiving the one or more analytes transferred bythe extraction system.
 28. The system of claim 27, wherein the systemcan be used to determine a concentration of the one or more analytes inthe substrate.
 29. The system of claim 28, wherein the step ofdetermining the concentration is done by an injection of an internalstandard, wherein the internal standard is a pure sample or a derivativeof the analyte having a similar mass.
 30. The system of claim 27,wherein the one or more analytes are selected from the group consistingof biomolecules, lipophilic compounds, oils, triacylglycerols, vegetableoil products, cottonseed oil, serum, proteins, antibodies, andcombinations and modifications thereof.
 31. The system of claim 30,wherein the oil is cottonseed oil.
 32. The system of claim 30, whereinthe serum is a rabbit serum.
 33. The system of claim 27, wherein themass spectrometer is equipped with a nanospray source.
 34. The system ofclaim 27, wherein the extraction solvent comprises water, polar organicand non-polar organic solvents, mixtures of polar and non-polarsolvents, and combinations and modifications thereof.
 35. The system ofclaim 27, wherein the extraction solvent is a 1:1 mixture of chloroformand methanol, comprising about 2% acetic acid.
 36. A method foridentifying lipid components, determining lipid profiles, detectingrelative amounts of different lipids, or any combinations thereof from alipid storing substrate, body, tissue, cell, organelle, lipid droplets(LDs) or any combinations thereof comprising the steps of: providing asample comprising the one or more lipid containing substrates, bodies,tissues, cells, organelles, LDs, or any combinations thereof; providingan extraction system for an extraction of the one or more lipids fromthe lipid containing tissue, cell, organelle, lipid droplets (LDs) orany combinations thereof followed by a transfer to a detection system,wherein the detection system may be coupled to the extraction system,wherein the extraction system comprises: a workstation comprising aplurality of moveable nanopositioners, wherein the nanopositioners arecapable of movement in a X direction and in a Y direction and in a Zdirection with respect to a fixed substrate, wherein the nanopositionershold one or more probes, grippers, capillary tips or any other suitableaccessory for an extraction and transfer of a liquid phase and thenanopositioners permit detection of analytes at the cellular orsubcellular level; a pressure injector for a delivery of a pressurizedextraction solvent through a capillary tip wherein the pressurizedextraction solvent contains at least one extraction solvent that isspecific for an analyte on the substrate that is to be dissolved,wherein the capillary tip is placed in close proximity to the lipidcontaining substrates, bodies, tissues, cells, organelles, LDs, or anycombinations thereof; a voltage source; a joystick or a digitalcontroller for controlling the movement of the nanopositioners; and amass spectrometer that is off line or is connected to the extractionsystem for receiving the one or more dissolved analytes transferred bythe extraction system; dissolving the one or more lipids by an injectionof the extraction solvent onto the substrate; aspirating the dissolvedlipids by the one or more capillary tips; injecting the aspirated lipidsinto the mass spectrometer to obtain a mass spectrum; and identifyingthe one or more lipids by a m/z ratio in the mass spectrum.
 37. Themethod of claim 36, further comprising the steps of: determining a lipidprofile from an extract of lipids or one or more lipid standards; andcomparing the identified lipids from the sample with the lipid profilefrom the extract of lipids or the lipid standards to determine the lipidprofile of the sample, the relative amounts of the lipids in the sample,or any combinations thereof.
 38. The method of claim 36, wherein themass spectrometer is equipped with a nanospray source.
 39. The method ofclaim 36, wherein the substrate is mounted on an inverted microscopicstage.
 40. The method of claim 36, wherein the extraction solventcomprises water, polar organic and inorganic solvents, mixtures of polarand non-polar solvents, and combinations and modifications thereof. 41.The method of claim 36, wherein the extraction solvent comprises 10 mMammonium acetate in chloroform:methanol (1:1, v/v).
 42. The method ofclaim 36, wherein the lipids comprise triacylglycerol (TAG),monoglycerides, diglycerides, triglycerides, lipid derivatives likefatty acid methyl esters (FAME), or any combinations thereof.
 43. Themethod of claim 36, further comprising the optional step of isolatingand purifying the lipid containing substrate, body, tissue, cell,organelle, LDs, or any combinations thereof.
 44. A method foridentifying lipids, determining lipid content, profile, or both or anycombinations thereof in a single-lipid droplet (LD) or directly from alipid containing organelle comprising the steps of: providing one ormore isolated and purified LDs or lipid containing organelle, whereinthe LDs or the lipid containing organelle are obtained from a plant oran animal source; providing an extraction system for an extraction ofthe LDs or the lipid containing organelle, followed by a transfer to adetection system, wherein the detection system may be coupled to theextraction system, wherein the extraction system comprises: aworkstation comprising a plurality of moveable nanopositioners, whereinthe nanopositioners are capable of movement in a X direction and in a Ydirection and in a Z direction with respect to a fixed substrate,wherein the nanopositioners hold one or more probes, grippers, capillarytips or any other suitable accessory for an extraction and transfer of aliquid phase and the nanopositioners permit detection of analytes at thecellular or subcellular level; a pressure injector for a delivery of apressurized extraction solvent through a capillary tip wherein thepressurized extraction solvent contains at least one extraction solventthat is specific for an analyte on the substrate that is to bedissolved, wherein the capillary tip is placed in close proximity to theLDs or the lipid containing organelle, wherein the extraction solventcomprises 10 mM ammonium acetate in chloroform:methanol (1:1, v/v)spiked with a Tri 15:0 triacylglycerol (TAG) standard; a voltage source;a joystick or a digital controller for controlling the movement of thenanopositioners; and a mass spectrometer that is off line or isconnected to the extraction system for receiving the one or more LDs orthe lipid containing organelle transferred by the extraction system;aspirating the LDs or the lipid containing organelle in the extractionsolvent by the one or more capillary tips; injecting the aspirated LDsor the lipid containing organelle into the mass spectrometer to obtain amass spectrum; and identifying the one or more lipids by a m/z ratio inthe mass spectrum.
 45. The method of claim 44, further comprising thesteps of: determining a lipid profile from an extract of lipids or oneor more lipid standards; and comparing the lipids from the LDs or thelipid containing organelle with the lipid profile from the extract oflipids or the lipid standards to determine the lipid profile of thesample, the relative amounts of the lipids in the sample, or anycombinations thereof.
 46. The method of claim 36, wherein the massspectrometer is equipped with a nanospray source.
 47. The method ofclaim 36, wherein the detection system may comprise a Ramanspectrometer.
 48. The method of claim 36, wherein the LDs or the lipidcontaining organelles may be stained using a lipid based dye or animaging agent.